Direct molecular cloning of foreign genes into poxviruses and methods for the preparation of recombinant proteins

ABSTRACT

A method is disclosed for producing a modified eukaryotic cytoplasmic DNA virus by direct molecular cloning of a modified DNA molecule comprising a modified cytoplasmic DNA virus genome. The inventive method comprises the steps of (I) modifying under extracellular conditions a DNA molecule comprising a first cytoplasmic DNA virus genome to produce a modified DNA molecule comprising the modified cytoplasmic DNA virus genome; (II) introducing the modified DNA molecule into a first host cell which packages the modified DNA molecule into infectious virions; and (III) recovering from the host cell virions comprised of the modified viral genome. The host cell is infected with a helper virus which is expressed to package the modified viral genome into infectious virions. Examples of packaging a modified poxvirus genome by a helper poxvirus of the same or different genus are described. Also disclosed are novel poxvirus vectors for direct molecular cloning of open reading frames into a restriction enzyme cleavage site that is unique in the vector. In one model poxvirus vector, the open reading frame is transcribed by a promoter located in the vector DNA upstream of a multiple cloning site comprised of several unique cleavage sites.

This is a continuation-in-part of U.S. Ser. No. 07/914,738, filed onJul. 20, 1992, now abandoned, which is a continuation-in-part of U.S.Ser. No. 07/750,080, filed Aug. 26, 1991, now U.S. Pat. No. 5,445,953.

BACKGROUND OF THE INVENTION

The present invention relates to modified genomes of eukaryotic DNAviruses which replicate in the cytoplasm of a host cell, such aspoxviruses and iridoviruses. More specifically, the invention relates todirect molecular cloning of a modified cytoplasmic DNA virus genome thatis produced by modifying under extracellular conditions a purified DNAmolecule comprising a cytoplasmic DNA virus genome. The modified DNAmolecule is then packaged into infectious virions in a cell infectedwith a helper cytoplasmic DNA virus. In a preferred embodiment of thepresent invention, a foreign DNA fragment comprising a desired gene isinserted directly into a genomic poxvirus DNA at a restrictionendonuclease cleavage site that is unique in the viral genome, and themodified viral DNA is packaged into virions by transfection into cellsinfected with a helper poxvirus.

Cytoplasmic DNA viruses of eukaryotes include diverse poxviruses andiridoviruses found in vertebrates and insects. Poxviruses havingrecombinant genomes have been used for expression of a variety ofinserted genes. Such poxviruses can be used to produce biologicallyactive polypeptides in cell cultures, for instance, and to delivervaccine antigens directly to an animal or a human immune system.Construction of recombinant iridovirus genomes for expression of foreigngenes appears not to be documented in the literature pertaining togenetic engineering.

Conventional techniques for construction of recombinant poxvirus genomescomprised of foreign genes rely in part on in vivo (intracellular)recombination. The use of intracellular recombination was firstdescribed as a process of “marker rescue” with subgenomic fragments ofviral DNA by Sam and Dumbell, Ann. Virol. (Institut Pasteur) 132E:135(1981). These authors demonstrated that a temperature-sensitive vacciniavirus mutant could be “rescued” by intracellular recombination with asubgenomic DNA fragment of a rabbit poxvirus. The methods they used forintracellular recombination are still used today.

Construction of recombinant vaccinia viruses comprised of non-poxvirus(“foreign”) genes was later described by Panicali and Paoletti, Proc.Nat'l Acad. Sci. U.S.A. 79:4927-4931 (1982); Mackett et al., Proc. Nat'lAcad. Sci. U.S.A. 79:7415-7419 (1982); and U.S. Pat. No. 4,769,330. Morespecifically, the extant technology for producing recombinant poxvirusesinvolves two steps. First, a DNA fragment is prepared that has regionsof homology to the poxvirus genome surrounding a foreign gene.Alternatively, an “insertion” plasmid is constructed by in vitro(extracellular) ligation of a foreign gene with a plasmid. This plasmidcomprises short viral DNA sequences that are homologous to the region ofthe poxvirus genome where gene insertion is ultimately desired. Theforeign gene is inserted into the plasmid at a site flanked by the viralDNA sequences and, typically, downstream of a poxvirus promoter thatwill control transcription of the inserted gene. In the second step, theinsertion plasmid is introduced into host cells infected with the targetpoxvirus. The gene is then indirectly inserted into the poxvirus genomeby intracellular recombination between homologous viral sequences in thepoxvirus genome and the portion of the plasmid including the foreigngene. The resulting recombinant genome then replicates, producinginfectious poxvirus.

Thus, insertion of each particular gene into a poxvirus genome hasheretofore required a distinct plasmid comprised of the gene flankedviral sequences selected for a desired insertion location. A difficultywith this approach is that a new insertion plasmid is required for eachrecombinant poxvirus. Each plasmid must be constructed by extracellularrecombinant DNA methods, amplified in a bacterial cell, and thenlaboriously isolated and rigorously purified before addition to apoxvirus-infected host cell.

Another problem with extant methodology in this regard is a low yield ofrecombinant genomes, which can necessitate screening hundreds ofindividual viruses to find a single desired recombinant. The poor yieldis a function of the low frequency of individual intracellularrecombination events, compounded by the requirement for multiple eventsof this sort to achieve integration of the insertion plasmid into aviral genome. As a result, the majority of viral genomes produced byintracellular recombination methods are parental genomes that lack aforeign gene. It is often necessary, therefore, to introduce a selectivemarker gene into a poxvirus genome, along with any other desiredsequence, to permit ready detection of the required rare recombinantswithout the need of characterizing isolated DNA's from numerousindividual virus clones.

Purified DNA's of eukaryotic cytoplasmic DNA viruses are incapable ofreplicating when introduced into susceptible host cells using methodsthat initiate infections with viral DNA's that replicate in the nucleus.This lack of infectivity of DNA's of cytoplasmic DNA viruses resultsfrom the fact that viral transcription must be initiated in infectedcells by a virus-specific RNA polymerase which is normally providedinside infecting virions.

“Reactivation” of poxvirus DNA, in which genomic DNA inside aninactivated, noninfectious poxvirus particle was packaged intoinfectious virions by coinfection with a viable helper poxvirus, hasbeen known for decades. See, for instance, Fenner and Woodroofe,Virology 11:185-201 (1960). In 1981 Sam and Dumbell demonstrated thatisolated, noninfectious genomic DNA of a first poxvirus could bepackaged into infectious poxvirus virions in cells infected with asecond, genetically distinct poxvirus. Sam and Dumbell, Ann. Virol.(Institut Pasteur) 132E:135 (1981). This packaging of naked poxvirus DNAwas first demonstrated by transfection of unmodified DNA comprising afirst wildtype orthopoxvirus genome, isolated from virions or infectedcells, into cells infected with a second naturally-occurringorthopoxvirus genome. However, heterologous packaging, packaging of DNAfrom one poxvirus genus (orthopox, for example) by viable virions ofanother genus (e.g., avipox), has not been demonstrated yet.

The use of intracellular recombination for constructing a recombinantpoxvirus genome expressing non-poxvirus genes was reported shortly afterSam and Dumbell first reported intracellular packaging of naked poxvirusDNA into poxvirus virions and marker rescue with DNA fragments byintracellular recombination. See Panicali and Paoletti, 1982; Mackett etal., 1982. The relevant literature of the succeeding decade, however,appears not to document the direct molecular cloning, i.e., constructionsolely by extracellular genetic engineering, of a modified genome of anyeukaryotic cytoplasmic DNA virus, particularly a poxvirus. Theliterature does not even evidence widespread recognition of anyadvantage possibly realized from such a direct cloning approach. To thecontrary, an authoritative treatise has stated that direct molecularcloning is not practical in the context of genetic engineering ofpoxviruses because poxvirus DNA is not infectious. F. Fenner et al., THEPOXVIRUSES. Academic Press, 1989). Others working in the area havelikewise discounted endonucleolytic cleavage and religation of poxvirusDNA's, even while recognizing a potential for rescue by infectious virusof isolated DNA comprising a recombinant poxvirus genome. See, forexample, Mackett and Smith, J. Gen. Virol. 67:2067-2082 (1986).Moreover, recent reviews propound the thesis that the only way feasibleto construct a recombinant poxvirus genome is by methods requiringintracellular recombination. See Miner and Hruby, TIBTECH 8:20-25(1990), and Moss and Flexner, Ann. Rev. Immunol. 5:305-324 (1987).

Vaccinia virus is a member of the Orthopox genus of the Poxvirus familywith little virulence for humans. Although the exact origin of vacciniavirus is obscure, it is related to the cowpox virus used by Jenner andstrains of vaccinia virus became the vaccines of choice for theprevention of smallpox. Baxby, “Vaccinia Virus,” in VACCINIA VIRUSES ASVECTORS FOR VACCINE ANTIGENS. G. V. Quinnan, ed., Elsevier, New York,N.Y., pp. 3-8 (1985). The smallpox vaccines used in the eradicationeffort were prepared on large scale by inoculating the shaved abdomensof calves, sheep or water buffalo with seed stocks of vaccinia virus andharvesting the infected exudative lymph from the inoculation sites.Henderson and Arita, “Utilization of Vaccine in the Global Eradicationof Smallpox,” VACCINIA VIRUSES AS VECTORS FOR VACCINE ANTIGENS. G. V.Quinnan, ed., Elsevier, New York, N.Y., pp. 61-67 (1985). The novelty ofthe vaccination procedure used by Jenner caused alarm with some of hiscontemporaries. The ultimate eradication of smallpox followingimplementation of the Intensified Smallpox Eradication Program of theWorld Health Organization proved that skepticism to be withoutfoundation.

Vaccinia virus has several biological properties which make it anexcellent candidate for use as a live vaccine. First, it possesses ahigh degree of physical and genetic stability under even severe fieldconditions, reducing problems and expense in transport and storage. Inaddition, genomic stability makes the incorporation of one or moreforeign genes for the antigens to be expressed more feasible than inother systems. Second, vaccinia replicates in the cytoplasm of hostcells and uses its own DNA and RNA polymerase. Its effects on the hostcell's physiologic functions can be minimized. Third, vaccinia virus hasa wide host range, thus permitting use of a single vaccine in a largenumber of species. Fourth, both humoral and cellular immunity aremediated by vaccinia virus-based vaccines. And fifth, the duration ofeffectiveness of vaccinia immunization is relatively long. See Haber etal., Science 243:51 (1989). Much of the early work geared towards avaccinia virus vector was undertaken with vaccine development in mind.Weir et al., Proc. Nat'l Acad. Sci. USA 79:1210-14 (1982); Mackett etal., Proc. Nat'l Acad. Sci. USA 79:7415-19 (1982); Smith et al., Nature302:490-95 (1983); Smith et al., Proc. Nat'l Acad. Sci. USA 80:7155-59(1983).

As with any vaccine, safety is a major concern with the use of vacciniavirus as a immunizing agent. The adverse reaction rate of 1 in 50,000,reported during smallpox vaccinations, was tolerated only because thedisease it prevented was so devastating. Baxby (1985). Generalizedvaccinia among persons without underlying illnesses is characterized bya vesicular rash of varying extent that is usually self-limited. In theevent of the formation of skin lesions as a result of virus replication,there is a risk of bacterial superinfection. In addition, there is alsoa risk of the formation of a scar at the site of skin lesions if theyoccur. Several attenuated smallpox vaccine strains were developed but,due to lower potency, were not adopted for general use. Recent effortstowards genetic engineering of vaccinia virus have resulted in strainswith decreased virulence. These efforts targeted the viral thymidinekinase, growth factor, hemagglutinin, 13.8 kD secreted protein andribonucleotide reductase genes. Buller et al., Nature 317:813 (1985);Buller et al., J. Virol. 62:866 (1988); Flexner et al., Nature 330:259(1987); Shida et al., J. Virol. 62:4474 (1988); Kotwal et al., Virology171:579 (1989); Child et al., Virology 174:626 (1990). There also isinterest in using other members of the poxvirus family, such asavipoxviruses, as limited host range vaccine vectors. Taylor et al.,Virology 6:497 (1988). For instance, U.S. Pat. No. 5,266,313, herebyincorporated by reference, discloses and claims a raccoon poxvirus-basedvaccine for rabies virus.

Recombinant vaccinia viruses have been used to express genes of nonviralpathogens such as bacteria, rickettsia and protozoa and, in some cases,have protected experimental animals from infection. Fields, Science252:1662-67 (1991). In addition, vaccinia-based rabies and rinderpestvaccines have been tested. Id. The human immunodeficiency virus type 1(HIV-1) envelope glycoprotein (env) gene has been cloned into a vacciniavector and a phase trial was conducted with this virus. The vaccineappeared safe, and demonstrated the development of readily detectable,persistent in vivo T-cell proliferative and serum antibody responses toHIV-1 in vaccinia-naive persons. Cooney et al., Lancet 337:567 (1991). Aneutralizing antibody response was not seen but the expression of theenv gene was low compared to levels now obtainable.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a methodfor constructing modified genomes of eukaryotic cytoplasmic DNA viruses,particularly of poxviruses, which overcomes the aforementionedlimitations associated with conventional techniques based onintracellular recombination.

It is another object of the present invention to provide cytoplasmic DNAvirus genome-construction techniques that produce substantially higheryields of recombinants than existing methodology.

It is a further object of this invention to provide methods formodifying a genome of a cytoplasmic DNA virus by direct modification ofgenomic viral DNA and intracellular packaging of the modified viral DNAinto virions with the aid of helper virus functions.

It is another object of this invention to provide methods forconstruction of a genome of a cytoplasmic DNA virus that produce in onerecombination reaction step modified genomes having a foreign DNAsegment inserted in each of the two possible orientations and modifiedgenomes having multiple insertions of a foreign DNA segment.

It is still another object of this invention to provide modified DNAmolecules suitable for direct molecular cloning of foreign genes in amodified cytoplasmic DNA virus genome, comprising two portions of agenomic viral DNA produced by cleavage with a sequence-specificendonuclease at a site that is unique in the viral genome.

It is yet a further object of this invention to provide a cytoplasmicDNA virus, particularly a poxvirus, having a modified genome comprisedof a foreign DNA inserted into a unique cleavage site for asequence-specific endonuclease.

It is another object of this invention to provide plasmids whichfacilitate construction and transfer of gene cassettes into acytoplasmic DNA virus, particularly a poxvirus, using direct molecularcloning.

It is yet another object of this invention to provide a cytoplasmic DNAvirus, particularly a poxvirus, having a modified genome comprised of aDNA encoding at least a portion of an HIV-1 antigen. Such a virus can beused to produce recombinant HIV-1 antigens in vitro.

It is another object of this invention to provide a subunit vaccineagainst HIV-1 infection using recombinant HIV-1 antigens generated usinga cytoplasmic DNA virus, particularly a poxvirus, having a modifiedgenome comprised of a DNA encoding at least a portion of an HIV-1antigen.

It is yet a further object of this invention to provide a cytoplasmicDNA virus, particularly a poxvirus, having a modified genome comprisedof a DNA encoding at least a portion of an HIV-1 antigen and suitablefor use as a live vaccine against HIV-1.

In accomplishing these and other objects, there has been provided, inaccordance with one aspect of the present invention, a method forproducing a modified eukaryotic cytoplasmic DNA virus by directmolecular cloning of a modified DNA molecule comprising a modifiedcytoplasmic DNA virus genome. The inventive method comprises the stepsof (I) modifying under extracellular conditions a purified DNA moleculecomprising a first cytoplasmic DNA virus genome to produce a modifiedDNA molecule comprising the modified viral genome; (II) introducing themodified DNA molecule into a first host cell which packages the modifiedDNA molecule into infectious virions; and (III) recovering from thefirst host cell infectious virions comprised of the modified viralgenome.

According to one embodiment of this method, the step of modifying theDNA molecule under extracellular conditions comprises a step of cleavingthe DNA molecule with a sequence-specific endonuclease. According toanother embodiment, the step of modifying the DNA molecule comprises astep of inserting a first DNA sequence into the first viral genome.Advantageously, this first DNA sequence is inserted into the firstgenome at a cleavage site for a sequence-specific endonuclease. Itshould be noted that where a particular sequence-specific endonuclease,such as a bacterial restriction enzyme, is described herein by name,that name also signifies any isoschizomer of the named nuclease.

Optionally, the step of modifying the DNA molecule according to thismethod also comprises a step of using a phosphatase to remove aphosphate moiety from an end of a DNA segment that is produced bycleaving the DNA molecule with a sequence-specific endonuclease.

In some embodiments of this method, the first viral genome is a vacciniavirus genome and the unique site is a cleavage site for the bacterialrestriction endonuclease NotI or for the bacterial restrictionendonuclease SmaI. The first genome also may comprise a second DNAsequence not naturally-occurring in a eukaryotic cytoplasmic DNA virusgenome where that second DNA sequence is comprised of the uniquecleavage site. For instance, the first genome may be a fowlpox virusgenome comprising a sequence of an Escherichia coli β-galactosidase geneand the unique site is a cleavage site for the bacterial restrictionendonuclease NotI that is located in that gene.

In other forms of this method, the first DNA sequence is inserted intothe first viral genome between a first cleavage site for a firstsequence-specific endonuclease and a second cleavage site for a secondsequence-specific endonuclease. Optionally, each of the first and secondcleavage sites is unique in the first viral genome.

According to other embodiments of the method of this invention, at leasta portion of the first DNA sequence which is inserted into the firstgenome is under transcriptional control of a promoter. This promoter maybe located in the first DNA sequence that is inserted into the firstviral genome. Alternatively, the promoter is located in the modifiedviral genome upstream of the first DNA sequence that is inserted intothe first genome. In some cases, the promoter is utilized by an RNApolymerase encoded by the modified viral genome. This promoter may alsobe suitable for initiation of transcription by an RNA polymerase of theeukaryotic cytoplasmic DNA virus to be modified. In certain methods, thepromoter comprises a modification of a naturally-occurring promoter ofthe eukaryotic cytoplasmic DNA virus.

The step of modifying the DNA molecule according to the method of thisinvention may comprise a step of deleting a DNA sequence from the firstgenome. Alternatively, this step comprises a step of substituting a DNAsequence of the first genome.

The method of modifying a first viral genome may also comprise a step ofinfecting the first host cell with a second eukaryotic cytoplasmic DNAvirus comprising a second genome which is expressed to package themodified viral genome into infectious virions. Advantageously, the stepof introducing the modified DNA molecule into the first host cell iscarried out about one hour after the step of infecting the first hostcell with the second eukaryotic cytoplasmic DNA virus.

In one variation of this method, the first host cell is selected suchthat expression of the second genome in the first host cell does notproduce infectious virions comprised of the second viral genome. Forinstance, where the modified viral genome is a modified vaccinia virusgenome and the second genome is a fowlpox virus genome, the selectedfirst host cell is a mammalian cell.

In some forms of the method of modifying a viral genome, the step ofrecovering infectious virions comprised of the modified viral genomecomprises a step of infecting a second host cell with infectious virionsproduced by the first host cell. This is done under conditions such thatexpression of the second genome in the second host cell does not produceinfectious virions comprised of the second genome. For instance, whenthe modified viral genome is a modified vaccinia virus genome, thesecond genome may be a fowlpox virus genome, and the second host cell isa mammalian cell. Alternatively, the modified viral genome comprises afunctional host range gene required to produce infectious virions in thesecond host cell and the second genome lacks that functional host rangegene. This is illustrated by the case where the modified viral genome isa modified vaccinia virus genome comprising a functional host range generequired to produce infectious virions in a human cell and the secondhost cell is a human cell.

In other forms of this method, the modified viral genome comprises aselective marker gene, the second genome lacks that selective markergene, and the step of infecting the second host cell is carried outunder conditions that select for a genome expressing that selectivemarker gene. Advantageously, expression of the selective marker gene inthe second host cell confers on the second host cell resistance to acytotoxic drug which is present during infection at a level sufficientto select for a genome expressing the selective marker gene.

According to another aspect of the present invention, there is provideda modified eukaryotic cytoplasmic DNA virus produced by direct molecularcloning of a modified viral genome according to methods summarizedhereinabove.

Yet another aspect of the present invention relates to a modifiedeukaryotic cytoplasmic DNA virus comprised of a modified viral genome,wherein that modified viral genome comprises: (I) a first genome of afirst eukaryotic cytoplasmic DNA virus. This first genome is comprisedof a cleavage site for a sequence-specific endonuclease and thiscleavage site is a unique site in the first genome. The modified genomefurther comprises (II) a first DNA sequence inserted into the uniquesite in the first genome.

According to a major embodiment of this aspect of the invention, thefirst DNA sequence is not naturally-occurring in a genome of aeukaryotic cytoplasmic DNA virus. In some preferred cases, the firstgenome is a vaccinia virus genome and the unique site is a cleavage sitefor a bacterial restriction endonuclease selected from the groupconsisting of NotI and SmaI.

The first genome may comprise a second DNA sequence notnaturally-occurring in a genome of a eukaryotic cytoplasmic DNA virusand that second DNA sequence is comprised of the unique cleavage site.In one example, the first genome is a fowlpox virus genome comprising asecond DNA sequence of an Escherichia coli β-galactosidase gene and theunique site in that gene is a cleavage site for the bacterialrestriction endonuclease NotI.

In some modified viruses of this invention, at least a portion of saidfirst DNA sequence that is inserted into the unique site is undertranscriptional control of a promoter. This promoter is located in thefirst DNA sequence that is inserted into the first genome. In some casesthe first genome is a poxvirus genome and the promoter comprises apoxvirus promoter, either a naturally-occurring poxvirus promoter or amodification thereof.

Yet another aspect of the present invention relates to a modifiedeukaryotic cytoplasmic DNA virus comprised of a modified viral genome inwhich the modified viral genome comprises (I) a first genome of a firsteukaryotic cytoplasmic DNA virus. This first genome is comprised of afirst cleavage site for a first sequence-specific endonuclease and asecond cleavage site for a second sequence-specific endonuclease. Eachof these cleavage sites is a unique site in the first genome.

The modified genome in this modified virus further comprises (II) afirst DNA sequence inserted into the first genome between the firstunique site and the second unique site. In some forms of this modifiedvirus the first DNA sequence is not naturally-occurring in a genome of aeukaryotic cytoplasmic DNA virus. In some cases the first genomecomprises a second DNA sequence not naturally-occurring in a genome of aeukaryotic cytoplasmic DNA virus and that second DNA sequence iscomprised of the first DNA sequence inserted between the first uniquesite and the second unique site. For an example, this modified virus maycomprise a first genome that is a vaccinia virus genome and each of thefirst unique site and the second unique site is a cleavage site for abacterial restriction endonuclease selected from the group consisting ofNotI, SmaI, ApaI and RsrII.

Yet another modified eukaryotic cytoplasmic DNA virus of the presentinvention is comprised of a modified viral genome which comprises (I) afirst genome of a first eukaryotic cytoplasmic DNA virus. This firstgenome is comprised of a first DNA sequence and this first DNA sequenceis comprised of a cleavage site for a sequence-specific endonucleasethat is a unique site in this modified viral genome. This genome of thismodified virus further comprises (II) a promoter located such that a DNAsequence inserted into the unique site in the viral genome is undertranscriptional control of the promoter. In certain forms, this firstDNA sequence lacks a translation start codon between the promoter andthe unique insertion site. This first DNA sequence may be one that isnot naturally-occurring in a genome of a eukaryotic cytoplasmic DNAvirus. This modified virus is exemplified by one in which the firstgenome is a vaccinia virus genome and the first DNA sequence iscomprised of a multiple cloning site comprising cleavage sites for thebacterial restriction endonucleases NotI, SmaI, ApaI and RsrII.

Yet another aspect of the present invention relates to a modifiedeukaryotic cytoplasmic DNA virus of this invention, wherein a firstsequence in the modified viral genome (an inserted sequence of interest)is expressed in a host cell resulting in production of a protein.

In one preferred embodiment of the foregoing aspect, the sequence ofinterest is derived from HIV-1, in particular, from the HIV-1 gp160, gagand pol genes. A cytoplasmic DNA virus containing such sequences isuseful for the production of recombinant HIV-1 antigens in tissueculture. Recombinant HIV-1 antigens can be used in subunit vaccines oras diagnostic agents. In addition, a cytoplasmic virus containing HIV-1sequences is useful as a live vaccine against HIV-1 infection.

According to another aspect, the present invention also relates to a DNAmolecule comprising a modified viral genome of a modified virusaccording to the present invention, In particular, some forms of thisDNA molecule comprise one end of a modified viral genome of a eukaryoticcytoplasmic DNA virus in which (I) that end of the modified viral genomecomprises a DNA sequence not naturally-occurring in a genome of aeukaryotic cytoplasmic DNA virus. In this DNA molecule, (II) themodified viral genome is comprised of a cleavage site for asequence-specific endonuclease that is a unique site in the modifiedviral genome; and (III) the DNA molecule has a terminus that ishomologous to a terminus that is produced by cleaving the unique site inthe modified viral genome with the sequence-specific endonuclease.

In some forms of this DNA molecule, the DNA sequence notnaturally-occurring in a genome of a eukaryotic cytoplasmic DNA virus iscomprised of the cleavage site for a sequence-specific endonuclease thatis a unique site in the modified viral genome.

Still another aspect of this invention relates to a kit for directmolecular cloning of a modified viral genome of a eukaryotic cytoplasmicDNA virus, comprising:

(I) purified DNA molecules according to this invention;

(II) a DNA ligase; and

(III) solutions of a buffer and reagents suitable for ligation of DNAsegments together to produce a modified DNA molecule comprising themodified viral genome. In one form, this kit further comprises a plasmidcomprised of a gene expression cassette flanked by sites for cleavagewith a sequence-specific endonuclease that are compatible for insertionof that cassette into a unique cleavage site of the modified viralgenome encoded by the DNA molecule in the kit. The kit may furthercomprise a first host cell and a second virus suitable for packaging ofthe modified viral genome into infectious virions.

According to a further aspect, this invention relates to a plasmidcomprising a DNA segment having a cleavage site for the bacterialrestriction endonuclease NotI at each end. In this plasmid, this DNAsegment comprises a sequence-specific endonuclease cleavage site that isunique in the plasmid. An example of this plasmid as shown in FIG. 1.3,is designated pN2. In this plasmid the DNA segment may further comprisea selective marker gene under transcriptional control of a poxviruspromoter. For instance, such plasmids include plasmids designatedpN2-gpta and pN2-gptb.

Another plasmid of the invention contains a DNA segment that furthercomprises a poxvirus promoter operatively linked to a DNA sequencecomprising a restriction endonuclease cleavage site. Thus, a DNA segmentinserted into this cleavage site is under transcriptional control ofthis promoter. Examples are plasmids designated pA1-S2 and pA2-S2. Anexample of such a plasmid which further comprises a selective markergene under control of a separate poxvirus promoter is plasmid pN2gpt-S4.

Still another plasmid comprises a segment of a poxvirus genome thatcomprises a thymidine kinase gene of that poxvirus. This thymidinekinase gene has been modified to prevent expression of active thymidinekinase, as in plasmids designated pHindJ-2 and pHindJ-3. Another plasmidcomprises a poxvirus promoter operatively linked to a translationalstart codon. This start codon is immediately followed by a secondrestriction endonuclease cleavage site suitably arranged to permittranslation of an open reading frame inserted into that secondrestriction endonuclease cleavage site. Examples of this plasmid includeplasmids designated pA1-S1, pA2-S1 and plasmid pN2gpt-S3A.

One particular plasmid of this type further comprises a DNA sequenceencoding human prothrombin, where that DNA sequence is operativelylinked to the poxvirus promoter and a start codon, as illustrated inFIG. 5.1 by a plasmid designated plasmid pA1S1-PT.

Another plasmid further comprises a DNA sequence encoding humanplasminogen and including a translation start codon, where that DNAsequence is operatively linked to the poxvirus promoter. As shown inFIG. 5.2, this is exemplified by plasmids derived from pN2gpt-S4, suchas pN2gpt-GPg, encoding human glu-plasminogen and pN2gpt-LPg encodinglys-plasminogen.

Yet another plasmid of this invention, as above, further comprises a DNAsequence encoding human immunodeficiency virus (HIV) gp160, including atranslation start codon, operatively linked to the poxvirus promoter, asshown in FIG. 5.4 by plasmid pN2gpt-gp160. Finally, another plasmidcomprises a DNA sequence encoding human von Willebrand factor as shownin FIG. 6.2, an example being designated plasmid pvWF.

Some plasmids of this invention comprise a sequence-specificendonuclease cleavage site that is unique in the genome of the poxvirus.Examples are shown in FIG. 4.3, including pA0, pA1 and pA2.

Another plasmid comprises a modified EcoRI K fragment of vaccinia virusDNA from which the K1L host range gene is deleted, as depicted in FIG.8.1. Two examples are pEcoK-dhr and pdhr-gpt.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.1 illustrates expression of marker genes by modified genomes ofpoxviruses produced by reactivation of naked poxvirus DNA. Asilver-stained polyacrylamide gel of proteins produced in culturesupernatants of cells infected with packaged viruses (vpPg#1-vpPg#8) andwith wildtype (WT) virus controls is shown. The upper arrow points toplasminogen marker band, the lower arrow, to the band of major secreted35 K vaccinia marker protein. Lanes 1 and 9, marker proteins; lanes 2and 10, human plasminogen standard (10 ng); lane 3, vaccinia recombinantvPgD (source of packaged DNA); lanes 4-7 and 11-14, vpPg#1-8; lanes 8and 15, wildtype vaccinia (WR WT).

FIG. 1.2 is a schematic diagram illustrating direct molecular cloning ofpoxvirus genomes comprised of a gene cassette for expression of a markergene (the E. coli gpt gene) under control of a vaccinia virus promoter.

FIG. 1.3 is a schematic illustration of construction of plasmids(pN2-gpta and pN2-gptb) which are precursors for construction of geneexpression cassettes by insertion of a promoter and an open readingframe. Such cassettes are designed for direct molecular transfer intovaccinia virus vectors using a unique insertion site and a selectablemarker gene (gpt) driven by a vaccinia virus promoter. MCS=multiplecloning site. P7.5=promoter for vaccinia 7.5K kDa polypeptide gene;P11=promoter for vaccinia 11K polypeptide gene. Arrows indicate thedirections of transcription from the promoters.

FIG. 1.4 demonstrates that poxvirus genomes produced by direct molecularcloning contain the gpt marker gene cassette inserted at a unique (NotI)cleavage site, as shown by Southern blot analyses of plaque-purifiedviral DNA's digested with the HindIII endonuclease using a gpt-geneprobe. Lane 1, marker DNA's (HindIII digested phage λ DNA); lanes 2 and3, wildtype vaccinia virus (WR) DNA cut with HindIII (500 and 100 ng,respectively); lanes 4-9, DNA's of cells infected with plaquesdesignated 2.1.1 through 7.1.1; lanes 10-12, DNA's of cells infectedwith plaques 10.1.1-12.1.1. Arrows indicate sizes of the restrictionfragments of the marker in kilobase pairs.

FIG. 1.5 further illustrates structures of modified poxvirus DNA's usingSouthern blot analyses of NotI-digested DNA's of cells infected withvarious isolates and hybridized with a gpt-gene probe. Lane 1, markerDNA's (HindIII digested phage λ DNA); lane 2, vaccinia wildtype (WT) DNAcut with NotI (50 ng); lanes 3-8, DNA's of cells infected withrecombinant plaques designated 2.1.1 through 7.1.1; lanes 9-11, DNA's ofcells infected with plaques 10.1.1-12.1.1.

FIG. 1.6 shows a comparison of DNA's from wildtype (WT) vaccinia and amodified clone (vp7) using ethidium bromide staining of DNA fragmentscleaved with indicated restriction endonucleases and separated on anagarose gel. Lanes 1 and 2, NotI digests of WT and vp7; lanes 3 and 4,HindIII digests of WT and vp7; lanes 5 and 6, HindIII and NotI combineddigests of WT and vp7; lanes 7 and 8, PstI digests of WT and vp7; lanes9 and 10, PstI and NotI combined digests of WT and vp7; lanes 11 and 12,SalI digests of WT and vp7; lane 13, marker DNA's (ligated and HindIIIdigested phage λ DNA; and phage øX cut with HaeIII). Arrows on the leftindicate sizes of fragments (in kilobase pairs) of NotI digest ofvaccinia WT; arrows on right, markers. Note that lanes 1 and 2 containabout tenfold less DNA than the other lanes.

FIG. 1.7 illustrates a Southern blot analysis of the gel shown in FIG.1.6 using a gpt-gene probe. Arrows indicate marker sizes.

FIG. 1.8 presents Southern blot analyses of vaccinia virus DNA's frominfected cells digested with NotI and hybridized to a vaccinia virusprobe. Lanes 1-4, DNA's of cells infected with plaques designated A1-A4;lanes 5-8, plaques C1-C4; lanes 9-12, plaques E1-E4; lane 13, vacciniaWT DNA; lane 14, DNA of uninfected CV-1 host cells; lane 15, markerDNA's (HindIII digested phage λ DNA; and phage øX cut with HaeIII).

FIG. 1.9 shows a Southern blot analysis of the same samples as in thegel shown in FIG. 1.8 using a gpt-gene probe. Lanes 1-12 as in FIG. 1.8;lane 13, DNA of uninfected CV-1 host cells; lane 14, vaccinia WT DNA;lane 15, marker DNA's (HindIII digested phage λ DNA; and phage øX cutwith HaeIII).

FIG. 1.10 shows a Southern blot analysis of the same viral DNA's as inthe gel in FIG. 1.8, restricted with PstI, using a gpt-gene probe. Lanes1-12 as in FIG. 1.8; lane 13, DNA of uninfected CV-1 host cells; lane14, vaccinia WT DNA; lane 15, marker DNA's (HindIII digested phage λDNA; and phage øX cut with HaeIII).

FIG. 1.11 outlines a schematic of the predicted structure of themodified PstI “C” fragments of vaccinia virus DNA's with single ordouble insertions of the gpt-gene cassette. P=PstI and N=NotI cleavagesites. The numbers indicates sizes of respective PstI fragments; boldtype numbers indicate fragments expected to hybridize with a gpt-geneprobe. Arrows indicate direction of transcription of the gpt-gene (800bp) by the vaccinia virus promoter (300 bp).

FIG. 2.1 presents analyses of recombinant avipox (fowlpox, FP) genomesby digestion with the restriction endonuclease NotI and separation byFIGE on a 1% agarose gel. Lane 5, marker (phage λ HindIII fragments,uncut phage λ and vaccinia WR); lanes 1 and 2, fowlpox virus HP1.441DNA, uncut and cut with NotI; lanes 3 and 4, recombinant fowlpox virusf-TK2a DNA, uncut and cut with NotI.

FIG. 2.2 illustrates construction of fowlpox viruses expressing foreigngenes by direct molecular cloning. A gene expression cassette,consisting of the E. coli gpt gene controlled by a poxvirus promoter (P)is ligated with the right and left DNA arms (ra and la, respectively) offowlpox virus (f-TK2a) obtained by cleavage with NotI. Packaging isperformed by fowlpox helper virus (strain HP2) in chicken embryofibroblasts.

FIG. 3.1 illustrates a process for construction of modified poxvirusesby extracellular genome engineering and intracellular packaging. A genecassette consisting of the gpt gene controlled by a vaccinia viruspromoter, is ligated with the “right arm” (ra) and the “left arm” (la)of vaccinia virus DNA cleaved at a unique site with the endonucleaseSmaI. Packaging is done by the fowlpox helper virus (strain HP1.441) inchicken embryo fibroblasts. P1=promoter of the vaccinia virus genecoding for the 7.5 kDA polypeptide.

FIG. 3.2 demonstrates that engineered vaccinia virus genomes packaged byfowlpox helper virus contain the expected insert at a unique SmaIcleavage site, as determined by Southern blot analyses. Total DNAisolated from infected cells was digested with HindIII, and the blot washybridized with a gpt-gene probe. Lanes 1-8, DNA's from cells infectedwith plaques designated F12.2-F12.9; lanes 9-13, plaques F13.1-F13.5;lanes 14 and 15, HindIII-digested DNA isolated from uninfected cells andcells infected with vaccinia (WR wildtype) virus, respectively; lane 16,markers (HindIII-digested phage λ DNA). The DNA in lane 8 does nothybridize because the virus isolate #F12.9 did not replicate.

FIG. 3.3 presents a schematic outline of the expected structures ofmodified vaccinia virus genomes having a gene cassette inserted into aunique SmaI site, particularly the modified HindIII “A” fragments ofviruses with single and double insertions. H=HindIII and S=SmaIrestriction endonuclease cleavage sites. Numbers indicate sizes of theHindIII fragments, with those in bold type indicating fragments expectedto hybridize with a gpt-gene probe. The gpt gene cassette consists of avaccinia virus promoter (about 300 bp in size) separated by an internalHindIII site from the gpt sequences (about 800 bp). Arrows indicate thedirection of transcription of the gpt gene.

FIG. 4.1A shows a schematic plan for the construction of vaccinia virusvector vdTK having a modified thymidine kinase (tk) gene using onlydirect molecular modification of the vaccinia virus genome, includingdeletion of undesired NotI and SmaI sites. WR-WT=wildtype (WT) WesternReserve (WR) strain of vaccinia virus (VV).

FIG. 4.1B outlines an alternative approach to that outlined in FIG. 4.1Afor deletion of a NotI site using marker rescue techniques with vacciniavirus and a modified plasmid.

FIG. 4.1C outlines an alternative method to that outlined in FIG. 4.1Afor deleting the SmaI site by marker rescue.

FIG. 4.2 illustrates construction of the vaccinia virus vector (vdTK)having the thymidine kinase (tk) gene replaced with a multiple cloningsite. The arrow indicates the initiation and direction of transcriptionof the vaccinia virus tk gene (VV-tk) in the HindIII J fragment clonedin plasmid pHindJ-1. The tk gene was replaced, as shown, and the finalplasmid pHindJ-3 was used to insert the modified HindIII J fragment intovaccinia virus.

FIG. 4.3 outlines construction of plasmids (pA1 and pA2) which areprecursors for construction of gene expression cassettes by insertion ofa promoter and an open reading frame. Such cassettes are suitable fordirect molecular transfer into vaccinia virus vector vdTK usingdirectional (forced) cloning.

FIG. 4.4A illustrates construction of plasmids (pA1-S1 and pA2-S1)comprised of gene expression cassettes suitable for association of openreading frames with a synthetic poxvirus promoter (S1) and a translationstart codon. The cassettes are designed for direct molecular transferinto vaccinia virus vector vdTK by forced cloning. The S1 promoter ispresent in different orientations in the two plasmids, as indicated bythe arrows showing the directions of transcription.

FIG. 4.4B shows the structure of the S1 promoter (bases 21-194 of SEQ IDNO:9).

FIG. 4.5A outlines the construction of plasmids (pA1-S2 and pA2-S2)comprised of gene expression cassettes suitable for association of openreading frames already having a translation start codon with a syntheticpoxvirus promoter (S2), prior to direct molecular transfer into vacciniavirus vector vdTK by forced cloning. The S2 promoter is present indifferent orientations in the two plasmids, as indicated by the arrowsshowing the directions of transcription.

FIG. 4.5B shows the structure of the S2 promoter (bases 21-73 of SEQ IDNO:11).

FIG. 4.6 shows the construction of plasmids pN2-gpta and pN2-gptb.

FIG. 4.7 shows construction of plasmids (pN2gpt-S3A and pN2gpt-S4)comprised of gene expression cassettes suitable for association of anopen reading frame, either lacking (S3A) or having (S4) a translationstart codon, with a synthetic promoter (S3A, bases 21-107 of SEQ IDNO:13, or S4, bases 21-114 of SEQ ID NO:14, respectively), prior todirect molecular transfer into a unique site in vaccinia virus vectorvdTK. Abbreviations as in FIG. 1.3.

FIG. 5.1 illustrates construction of a gene expression cassette plasmid(pA1S1-PT) for expression of human prothrombin in vaccinia virus vectorvdTK. Abbreviations as in FIG. 1.3. Arrows indicate the direction oftranscription.

FIG. 5.2 presents construction of a gene expression cassette plasmid(pN2gpt-GPg) for expression of human glu-plasminogen in vaccinia virusvector vdTK. S4=synthetic poxvirus promoter; other abbreviations as inFIG. 1.3.

FIG. 5.3 shows construction of a gene expression cassette plasmid(pN2gpt-LPg) for expression of human lys-plasminogen in vaccinia virusvector vdTK. Abbreviations as in FIG. 1.3.

FIG. 5.4 outlines construction of a gene expression cassette plasmid(pN2gpt-gp160) for expression of a human virus antigen (HIV gp160) invaccinia virus vector vdTK. Abbreviations as in FIG. 1.3.

FIG. 5.5 illustrates an approach for screening of modified vacciniaviruses made by direct molecular cloning based on concurrent insertionof a marker gene (the E. coli lacZ gene) which confers a visuallydistinctive phenotype (“blue” plaque compared to normal “white” plaquesof viruses lacking a lacZ gene).

FIG. 5.6 illustrates the construction of plasmids pTZS4-lacZa andpTZS4-lacZb.

FIG. 6.1 illustrates construction of a vaccinia virus vector (vS4) witha directional master cloning site under control of a strong latevaccinia virus promoter (S4). SEQ ID NOS 38 and 39 are shown in thisFigure.

FIG. 6.2 presents construction of a modified vaccinia virus (vvwF) forexpression of von-Willebrand factor by direct molecular insertion of anopen reading frame into vaccinia virus vector vS4. vWF=von Willebrandfactor cDNA. The arrow indicates the direction of transcription from theS4 promoter.

FIG. 7.1 illustrates the effect of amount of added DNA on packaging ofvaccinia virus DNA by fowlpox helper virus in mammalian (CV-1) cells inwhich fowlpox virus does not completely replicate. Five cultures wereinfected with fowlpox virus and subsequently transfected with theindicated amounts of vaccinia virus DNA. The first column indicates aculture with no added DNA and no fowlpox virus, and the fifth column, noadded DNA but infected with fowlpox virus.

FIG. 8.1 outlines construction of a vaccinia virus (vdhr) suitable foruse as a helper virus having host range mutations which preventreplication in some human cell lines. hr-gene=host range gene located inthe EcoRI K fragment of vaccinia virus; other abbreviations as in FIG.1.3.

FIG. 9.1 shows the construction of the plasmids pS2gpt-P2 andpP2gp160MN. Arrows within plasmids show the direction of transcriptionof the respective genes. SEQ ID NO:43 is shown in this Figure.

FIG. 9.2 shows the schematic outline of the construction of the virusesvP2-gp160MN-A and vP2gp160MN-B.

FIG. 9.3 shows maps of the PstI-E-fragment of the wild-type vacciniavirus and of the PstI-fragments of the chimeric viruses comprising gp160genes. Arrows indicate the direction of transcription of the gp160 gene.Numbers indicate sizes of fragments in kilobase pairs.

FIG. 9.4 shows construction of the plasmid pselP-gpt-L2. Arrows indicatethe direction of transcription of the respective genes. SEQ ID NO:86 isshown in this Figure.

FIG. 9.5A shows construction of the plasmid pselP-gp160MN. SEQ ID NO:86is shown in this Figure.

FIG. 9.5B shows sequences around translational start codons of wild-type(SEQ ID NOS 73 and 74) and modified (SEQ ID NOS 75 and 76) gp160 genes.

FIG. 9.6 is a schematic outline of construction of the chimeric vacciniaviruses vselP-gp160MNA and vP2-gp160MNB. Arrows indicate the directionof transcription of the gp160 gene.

FIG. 9.7 is a map of the SaII-F-fragment of the wild-type vaccinia virusand of SaII-fragments of chimeric vaccinia viruses vselP-gp160MNA andvP2-gp160MNB. Arrows indicate the direction of transcription of thegp160 gene. Numbers indicate sizes of the fragments in kilobase pairs.

FIG. 10.1A shows the structure of plasmid pN2-gptaProtS. The double genecassette consisting of the gpt gene controlled by the vaccinia P7.5promoter (P7.5) and the human Protein S gene (huProtS) controlled by asynthetic poxvirus promoter (selP) is flanked by NotI restriction sites.

FIG. 10.1B shows sequences around translational start codons ofwild-type Protein S gene (SEQ ID NOS 77 and 78) and the Protein S genein the chimeras (SEQ ID NOS 79 and 80).

FIG. 10.2A shows Southern blot analysis of chimeric vaccinia virusescarrying the Protein S gene. Total cellular DNAs digested with SacI andhybridized with vaccinia wild-type SacI fragment.

FIG. 10.2B shows the same material of FIG. 10.2A digested with NotI andprobed with human Protein S sequences.

FIG. 10.2C shows schematic outline of the wild-type SacI-I-fragment andthe chimeric SacI-fragment after ligation of the insert.

FIG. 10.3 shows Western blot analysis of plasma-derived Protein S(pdProtS; lanes 1 and 2) and of recombinant Protein S (rProtS). Cellculture supernatants (10 μl) of SK Hep1 cells were assayed afterincubation periods of 24-72 h.

FIG. 11.1A shows the structure of plasmid pN2gpta-FIX. The double genecassette consisting of the gpt gene controlled by the vaccinia P7.5promoter (P7.5) and the human factor IX gene controlled by a syntheticpoxvirus promoter (selP) is flanked by NotI restriction sites.

FIG. 11.1B shows sequences from wild-type factor IX (SEQ ID NOS 81 and82) and factor IX vFIX#5 (SEQ ID NOS 83 and 84).

FIG. 11.2A shows Southern blot analysis of the chimeric viruses carryinga gene for human factor IX. Total cellular DNAs digested with SfuI andhybridized with the human factor IX gene probe (plasmidpBluescript-FIX). In all eight isolates (#1-6, 9 and 10), the insert hadthe ‘a’-orientation. m=marker; VV-WT/WR=vaccinia wild-type, WR-strain.

FIG. 11.2B shows predicted genomic structures of the chimeric viruses.

FIG. 11.3 shows Western blot analysis of plasma-derived factor IX(pdFIX; lanes 1 and 2) and of recombinant factor IX expressed bychimeric vaccinia virus in Vero cells. Cell culture supernatants (10 μl)were assayed after incubation for 72 h. #1-6, 9 and 10=numbers of plaqueisolates; pd FIX=plasma-derived factor IX.

FIG. 12.1 illustrates construction of the chimeric fowlpox virusf-envIIIB by direct molecular cloning of and HIV-_(IIIB) env gene.

FIG. 12.2A shows Southern blot analyses of SspI-fragments of chimericfowlpox virus isolates showing orientations of env gene inserts. Lanes1-12, viral isolates f-LFa-l; lane 13 and 14, HP1.441 and f-TK2a(negative controls); lane 15, SspI digest (10 ng) of pN2gpt-gp160(positive control).

FIG. 12.2B shows restriction maps of SspI fragments of inserts in thetwo possible orientations in the chimeric fowlpox virus of FIG. 12.2A.Numbers indicate sizes of SspI-fragments in kilobase pairs. Arrowsindicate orientations of the insert which coincide with direction oftranscription of the gp160 transcription unit.

FIG. 12.3 shows expression of HIV-1 envelope glycoproteins in chickenembryo fibroblasts (Western blot analysis). Lanes 1-8, viral isolatesf-LF2a-h; lanes 9 and 15, gp160 standard, provided by A. Mitterer,Immuno Ag, Orth/Donau, Austria; lanes 10-13, viral isolates f-lF2i-l;lane 14, marker proteins; lanes 16 and 17, fowlpox viruses HP1.441 andf-TK2a (negative controls).

FIG. 12.4 shows detection of HIV gp41 produced by chimeric vacciniaviruses in infected chicken embryo fibroblasts (Western blot analysis).Lanes 1-8, viral isolates f-LF2a-h; lanes 9 and 15, gp160 standard;lanes 10-13, viral isolates f-LF2i-l; lane 14, marker proteins; lanes 16and 17, fowlpox viruses HP1.441 and f-TK2a (negative controls).

FIG. 13.1 shows the construction scheme of the plasmid pSep-ST2.

FIG. 13.2 shows the structure, sequence and construction of thesemi-synthetic poxvirus promoter Sep. SEQ ID NOS 87-89 are shown in thisFigure.

FIG. 13.3 shows a Southern blot analysis of viral genomic DNA of theviruses vRMN6b1 and the Western Reserve wild-type strain (WR-WT).

FIG. 13.4 shows a Southern blot analysis of viral genomic DNA of theviruses vRMN6b1 and the Western Reserve wild-type strain (WR-WT).

FIG. 13.5 shows a comparison of the gpl6OMN expression levels of thedifferent viral isolates.

FIG. 13.6 shows a comparison of gpl6OMN expression of vaccinia Sep-gpl6Oconstructs and vaccinia/bacterlophage T7 promoter constructs in CV-1 andVero cells. The arrow points to the gpl6O protein band.

FIG. 14.1 shows the construction of the plasmid pSep-gag. Sep is ahybrid, early/late promoter; P7.5 is the vaccinia promoter for the P7.5kDa product. Arrows indicate the direction of transcription. SEQ ID NOS94 and 95 are shown in this Figure.

FIG. 14.2 shows screening of viral crude stocks by Western blotanalysis. Confluent CV-1 cells were infected with 1 plaque forming unitof the respective virus and cultured for 48 hours. Total cellularproteins were separated on a 12% polyacrylamide gel and analyzed byWestern blot analysis. The protein samples of vgagl.l, vgagl.2 vgag2. 1,vgag2.2 and vgag7.1 were loaded in duplicate. The positive control,VVKI, is a gag/pol gene containing vaccinia recombinant described byKaracostas et al. (1989). WR-WT is the Western Reserve wild-type strain.HIV-IMN/H9 are total cellular proteins of HIV-lMN-infected H9 cells.CV-1 non are uninfected CV-1 cells. The arrow at the right sideindicates the p55 gag precursor protein.

FIG. 14.3 shows Southern blot analyses of HIV-1 gag-expressing viruseshybridized to the gag gene probe. The marker (m) consisted of phagelambda HindIII and phage phi X HaeIII fragments (the fragment sizes areshown kilobase pairs on the right side). WR-WT is the Western Reservewild-type strain to which the probe does not hybridize. Total DNAextracted from non-infected CV-1 cells (CV-1 DNA) served as a negativecontrol. Positive hybridization controls were HindIII (H) and Asp718 (A)fragments of the plasmid psep-gag.

FIG. 14.4 shows Southern blot analyses of HIV-1 gag-expressing viruseshybridized to the Not region probe. For abbreviations, see the legend ofFIG. 14.3.

FIG. 14.5 shows a comparison of the gag protein expression on CV-1 anVero cells. Confluent CV-1 or Vero cells were infected with 1 plaqueforming unit of the respective viruses and grown for 72 hours. Totalcellular proteins were analyzed as described in the legend of FIG. 14.2.The arrow at the right side points to the p55 gag precursor protein.

FIG. 15.1 shows construction of the plasmid pSep-gagpolIIIB. Arrowsaround plasmids indicate the direction of transcription of therespective gene cassettes. The numbers near the restriction endonucleasecleavage site indicate the positions of the sites in the respectiveplasmids. SEQ ID NOS 94 and 95 are shown in this Figure.

FIG. 15.2 shows screening of viral crude stocks by Western blotanalysis. Confluent CV-1 cells were infected with 3 plaque forming unitsof the respective virus and grown for 48 hours. Total cellular proteinswere separated on a 12% polyacrylamide gel and analyzed by Western blotanalysis. The protein samples of vgagpol 7, vgagpol 9 and vgagpol 10were loaded in duplicate. VVKI is a gag-pol gene containing the vacciniarecombinant described by Karakostas et al. (1989). WR-WT is the WesternReserve wild-type strain. HIV-I/H9 are total cellular proteins ofHIV-1-infected H9 cells. The arrows at the right side point to p55 gagand pl60 gag-pol precursor proteins.

FIG. 15.3 shows Southern blot analyses of HIV-1 gag-pol expressingviruses hybridized to the gag-pol gene probe. The size markers consistedof the Asp7l8 (ml) and HindIII (m2) fragments of the plasmidpSep-gagpolIIIB (the numbers on the right side are the fragment sizes inkilobase pairs). WR-WT is the Western Reserve wild-type strain to whichthe probe does not hybridize.

FIG. 16.1 shows maps of the PstI fragments of the fowlpox virus strainHPI.441, the strain f-TK2a and of both possible orientations of thechimeric viruses f-aMN (‘a’- and ‘b’orientation). FPV-tk=fowlpoxvirusthymidine kinase gene; 3′-orf=downstream open reading frame;VV-tk=vaccinia thymidine kinase gene; lacZ=E. coli β-galactosidase;gpt=E. coli xanthine guanine phosphoribosyl transferase, The arrowsindicate the direction of transcription.

FIG. 16.2 shows Western blot analysis of total chicken embryo fibroblast(CEF) proteins infected with different fowlpox viruses. The f-aMNchimeric viruses were three times plaque purified. The gpl6OMN standardwas purified from vaccinia virus-infected Vero cells. HP1.441 is theattenuated fowlpox virus strain from which f-TK2a was derived. CEF mockare total cellular proteins of a mock infection of chicken embryofibroblasts.

FIG. 16.3 shows Western blot analysis of total proteins from CV-1 andVero cells infected with the fowlpox virus f-aMN. The gpl6OMN standardwas purified from vaccinia virus-infected Vero cells. ‘Vero mock’ aretotal cellular proteins of a mock infection of Vero cells. The sampleswere applied in duplicate.

FIG. 16.4 shows Southern blot analyses of HIV-1 gpl6OMN-expressingfowlpox viruses hybridized to the gpl6O gene probe. The size markersconsisted of the HindIII fragments of the plasmid pSep-ST2 (pSep-ST2 H)(numbers on the right side are the sizes in kilobase pairs). Thewild-type strains HP1.441 and f-TK2a do not hybridize with the probe.

DETAILED DESCRIPTION OF THE INVENTION

The present invention represents the first construction of a modifiedgenome of a eukaryotic cytoplasmic DNA virus, as exemplified by apoxvirus, completely outside the confines of a living cell. Thisconstruction was accomplished using an isolated viral genomic DNA thatwas cleaved by a sequence-specific endonuclease and then religated withforeign DNA. The resulting modified DNA was then packaged intoinfectious poxvirus virions by transfection into a host cell infectedwith another poxvirus that served as a helper virus.

The present invention enables diverse strategies for vector developmentfrom eukaryotic cytoplasmic DNA viruses which have been appliedpreviously to other DNA viruses to solve various genetic engineeringproblems. For instance, this direct cloning approach offers thepossibility of cloning genes directly in cytoplasmic DNA viruses, suchas poxviruses, that cannot be cloned in bacterial systems, eitherbecause they are too large for bacterial vectors or are toxic tobacteria or are unstable in bacteria. Direct molecular cloning allowsgreater precision over construction of engineered viral genomes andunder optimum conditions can increase the speed of cloning as well asproduce a variety of constructs in a single ligation reaction, havingmultiple inserts in various orientations, which permits rapid screeningfor arrangements affording optimal expression of a foreign gene.

As used in the present context, “eukaryotic cytoplasmic DNA virus”includes iridoviruses and poxviruses. “Iridovirus” includes any virusthat is classified as a member of the family Iridoviridae, asexemplified by the African swine fever virus as well as certainamphibian and insect viruses. “Poxvirus” includes any member of thefamily Poxviridae, including the subfamililes Chordopoxviridae(vertebrate poxviruses) and Entomopoxviridae (insect poxviruses). See,for example, B. Moss in VIROLOGY, ed. Fields et al., Raven Press (1990)p. 2080. The chordopoxviruses comprise, inter alia, the following generafrom which particular examples are discussed herein, as indicated inparentheses: Orthopoxvirus (vaccinia); Avipoxvirus (fowlpox);Capripoxvirus (sheeppox) Leporipoxvirus (rabbit (Shope) fibroma,myxoma); and Suipoxvirus (swinepox). The entomopoxviruses comprise threegenera designated A, B and C.

According to one aspect of the present invention, a method is providedfor producing a modified eukaryotic cytoplasmic DNA virus by directmolecular cloning of a modified cytoplasmic DNA virus genome. Thismethod comprises a step of modifying under extracellular conditions apurified DNA molecule comprising a first cytoplasmic DNA virus genome toproduce a modified DNA molecule comprising a modified cytoplasmic DNAvirus genome.

A purified DNA molecule suitable for modification according to thepresent method is prepared, for example, by isolation of genomic DNAfrom virus particles, according to standard methods for isolation ofgenomic DNA from eukaryotic cytoplasmic DNA viruses. See, for instance,Example 1, hereinbelow. Alternatively, some or all of the purified DNAmolecule may be prepared by molecular cloning or chemical synthesis.

Modifying a purified DNA molecule comprising a virus genome within thescope of the present invention includes making any heritable change inthe DNA sequence of that genome. Such changes include, for example,inserting a DNA sequence into that genome, deleting a DNA sequence fromthat genome, or substitution of a DNA sequence in that genome with adifferent DNA sequence. The DNA sequence that is inserted, deleted orsubstituted is comprised of a single DNA base pair or more than one DNAbase pair.

According to this aspect of the invention, the step of modifying a DNAmolecule comprising a first DNA virus genome is performed with anytechnique that is suitable for extracellularly modifying the sequence ofa DNA molecule. For instance, modifying a DNA molecule according to thepresent invention comprehends modifying the purified DNA molecule with aphysical mutagen, such as ultraviolet light, or with a chemical mutagen.Numerous methods of extracellular mutagenesis of purified DNA moleculesare well known in the field of genetic engineering.

In another embodiment, the step of modifying the DNA molecule comprisesjoining together DNA segments to form the modified DNA molecule whichcomprises the modified viral genome. According to one aspect of thisembodiment, some or all of the DNA segments joined together to form themodified DNA molecule are produced by cleaving the DNA moleculecomprising the first virus genome with a nuclease, preferably asequence-specific endonuclease. Alternatively, some or all of the DNAsegments joined together to form the modified DNA molecule may beproduced by chemical synthesis using well known methods.

In some embodiments, the step of joining together DNA segments toproduce the modified DNA molecule comprises an extracellular step ofligating those DNA segments together using a ligase, such as a bacterialor bacteriophage ligase, according to widely known recombinant DNAmethods. optionally, this DNA modification step also comprises treatingends of DNA segments cleaved from the DNA molecule comprising the firstvirus genome with a phosphatase, for instance, calf intestinephosphatase. This enzyme removes phosphate moieties and thereby preventsreligation of one DNA segment produced by cleaving the DNA molecule withanother such segment.

In an alternative approach to joining the DNA segments, some or all ofthe DNA segments are joined by extracellular annealing of cohesive endsthat are sufficiently long to enable transfection of the modified DNAmolecule into a host cell where ligation of the annealed DNA segmentsoccurs.

In another embodiment of this method, the step of modifying the DNAmolecule comprising the first virus genome includes a step of joining atleast some DNA segments resulting from cleaving a genomic DNA moleculeof the first virus together with an additional DNA segment to producethe modified DNA molecule. In a preferred embodiment of this aspect ofthe invention, this step comprises cleaving a genomic viral DNA moleculewith a sequence-specific endonuclease at a unique cleavage site in thefirst virus genome, thereby producing two DNA “arms” of the genomicvirus DNA. The two arms are then ligated together with a foreign DNAcomprising a sequence of interest.

A DNA sequence of interest as used herein to describe the sequence of aforeign DNA segment that is ligated with virus DNA arms comprises, inthe first instance, a DNA sequence that is not naturally-occurring in agenome of a eukaryotic cytoplasmic DNA virus. Alternatively, a DNAsequence of interest comprises a sequence comprised of a sequence thatis naturally-occurring in a genome of a eukaryotic cytoplasmic DNA virusas well as a sequence that is not naturally-occurring in such a genome.Furthermore, a sequence of interest may comprise only sequences that arenaturally-occurring in a eukaryotic cytoplasmic DNA virus, where such asequence is inserted into a location in the genome of that cytoplasmicDNA virus different from the location where that sequence naturallyoccurs. Moreover, insertion of a naturally-occurring viral sequence ofinterest from one DNA virus into another, or from one part of a singleviral genome into another part of that genome, will necessarily create asequence that is “not naturally-occurring in the genome of a cytoplasmicDNA virus” according to the present invention, at the junction of theviral genome and the inserted viral sequence of interest.

The foreign DNA segment that is ligated to the two arms of genomic virusDNA comprises ends that are compatible for ligation with the ends of theviral DNA arms. The compatible ends may be complementary cohesive endsor blunt ends. The ligation step in this particular method produces amodified DNA molecule comprising the first virus genome with the DNAsequence of the foreign DNA inserted into the first virus genome at theunique cleavage site.

This embodiment of a method in which a DNA sequence is inserted into thegenome of the first virus is exemplified herein by, inter alia, a methodfor inserting a gene expression cassette into a vaccinia virus genome ata unique cleavage site for the bacterial restriction endonuclease NotIor SmaI, as described in Examples 1 and 3, respectively. This embodimentis also exemplified by insertion of a gene cassette into the genome of arecombinant fowlpox virus vector, at a unique NotI site within thesequence of a bacterial gene within the recombinant fowlpox virusgenome, as described in Example 2.

Inserting a foreign DNA into a unique site in a eukaryotic cytoplasmicDNA virus genome according to the present invention is useful for thepurpose of expressing a desired protein, particularly a human protein.For instance, Example 5 describes insertion of genes for plasminogen,prothrombin and human immunodeficiency virus glycoprotein 160 (HIVgp160) into a unique cleavage site of a vaccinia virus vector and theuse of the resulting modified vaccinia viruses for production of theseproteins. The foreign proteins may be produced in cell cultures, forpreparing purified proteins, or directly in human or animal hosts, forimmunizing the host with a vaccine comprising a modified virus accordingto the present invention.

In certain embodiments, the step of modifying a virus genome byinserting a DNA sequence comprises introducing or eliminating a markergene function for distinguishing the modified virus genome from thefirst virus genome. In one such embodiment, a DNA sequence inserted intothe first virus genome comprises a selective marker gene and the step ofrecovering the infectious modified poxvirus virions produced by thefirst host cell comprises a step of infecting a second host cell withthose infectious virions under conditions that select for a poxvirusgenome expressing the selective marker gene. In a preferred embodimentof this aspect of the invention, expression of the selective marker genein the second host cell confers on the second host cell resistance to acytotoxic drug. This drug is present during infection of the second hostcell at a level sufficient to select for a poxvirus genome expressingthe selective marker gene. In this case the drug selects for a modifiedvirus genome having the inserted selective marker gene and selectsagainst any genome lacking that marker gene.

Insertion of a DNA sequence comprising a selective marker gene fordistinguishing the modified virus genome from the first virus genome isparticularly useful when a genomic DNA molecule of the first virus hasbeen cleaved at a unique cleavage site and, therefore, the resultingviral DNA arms are likely to religate without insertion of the desiredDNA sequence. This approach is exemplified by a method for inserting agene for the enzyme xanthine-guanine-phosphoribosyl-transferase ofEscherichia coli (hereinafter, the “gpt” gene) into, inter alia, avaccinia virus genome or a fowlpox virus genome at a unique NotI site,as described in Examples 1 and 2, respectively.

A method for eliminating a marker gene function from the first virusgenome to distinguish the modified viral genome from the first genome isexemplified in Example 2. This method relates to insertion of a foreignDNA sequence into a fowlpox virus genome into a NotI site residing in anE. coli lacZ gene coding for β-galactosidase. As described in Example 2(avipox), insertion of a DNA sequence into this site disrupts the lacZcoding sequence and thereby prevents production of β-galactosidase.Expression of this enzyme produces a “blue plaque” phenotype for a viruscarrying the lacZ gene. Accordingly, a modified viral genome carrying aninsertion of a DNA sequence in this site exhibits a white plaquephenotype that distinguishes the modified virus from the first virus. Inother embodiments of methods according to this invention, a functioningE. coli lacz gene is transferred into the vector with another gene ofinterest to serve as a marker for modified viruses containing thedesired insert.

In still other embodiments of the method of this invention, the step ofmodifying a DNA molecule comprises introducing a new cleavage site for asequence-specific endonuclease into the first virus genome. One exampleof this embodiment comprises inserting into a existing unique site in afirst poxvirus genome a foreign DNA comprised of a synthetic DNA“linker”, as described in Example 6. This linker comprises a “multiplecloning site” comprised of several closely adjacent cleavage sites thatare useful for insertion of foreign DNA into the modified poxvirusgenome. Advantageously, the cleavage sites in the multiple cloning siteare not present in the first viral genome and, therefore, are unique inthe modified viral genome.

More particularly, the step of modifying a DNA molecule comprising afirst viral genome also includes inserting a DNA sequence between afirst and a second cleavage site for a sequence-specific endonuclease.In one such embodiment, the first viral genome comprises a multiplecloning site comprised of cleavage sites that are unique in the firstviral genome. According to this method, cleaving a DNA moleculecomprising a first viral genome at two such unique sites in the multiplecloning site produces two viral DNA arms having cohesive ends that arenot compatible for ligation with each other. The intervening DNA segmentbetween the two unique cleavage sites in the multiple cloning site isremoved from the cleaved viral DNA arms, for example, by ethanolprecipitation of these arms, as described for inserting a humanprothrombin gene into a modified poxvirus vector in Example 5.

Inserting a DNA segment into a viral genome between two unique cleavagesites is useful for “forced” cloning of DNA inserts having cohesive endscompatible for ligation with each of the vector arms. In other words,this method involving cleavage of viral DNA at two sites is useful forincreasing the yield of viral genomes resulting from ligation of viralDNA arms compared to arms prepared by cleavage of viral DNA at a singlesite, because the arms of this method do not have ends compatible forligation. This forced cloning method also directs orientation of the DNAinserted within the modified viral genome because only one viral DNA armis compatible for ligation to each end of the inserted DNA.

The forced cloning method of the present invention is demonstrated, forexample, by insertion of a gene expression cassette comprised of a humanprothrombin gene into a multiple cloning site of a vaccinia virusvector, as described in Example 5.

In a preferred embodiment, the intervening DNA segment between twounique cleavage sites in the first viral genome is not essential forreplication of the first viral genome and, therefore, neither deletingthis sequence nor replacing it with another DNA segment preventsreplication of the resulting modified genome. Alternatively, theintervening DNA segment is replaced by a DNA segment comprising thatportion of the intervening sequence that is essential for viralreplication linked to an additional DNA sequence that is to be insertedinto the first viral genome.

In another aspect of the present method, the step of modifying the firstviral genome comprises eliminating an undesirable cleavage site for asequence-specific endonuclease. Modifications of this type can be maderepeatedly, if necessary, for example, to delete redundant cleavagesites for the same nuclease, thereby ultimately producing a modifiedviral genome having a unique cleavage site for a particular nuclease.

Methods that are particularly suitable for eliminating a cleavage sitefrom a viral genome are known in the art. These include various generalsite-specific mutagenesis methods. One particular method for eliminatingan endonuclease cleavage site from a viral genome involves extracellulartreatment of genomic viral DNA to select for mutant genomic DNAmolecules that are resistant to cleavage by the pertinent endonuclease.

Another method for eliminating a cleavage site from a viral genome is byligating a cleaved viral DNA molecule with a DNA segment, for instance,a synthetic DNA segment, comprising an end compatible for ligation withthe cleaved viral DNA but lacking a portion of the recognition sequencefor the nuclease that cleaved the viral DNA. In this method, thecleavage site for the sequence-specific endonuclease that cleaves theviral DNA comprises a nuclease recognition sequence that extends beyondthe sequences encompassed in the cohesive ends into the sequencesimmediately adjacent to the cohesive ends. The synthetic insertcomprises cohesive ends compatible for ligation with the viral DNA armscleaved at a single site. However, the sequence immediately adjacent toone cohesive end of the synthetic insert differs from the recognitionsequence that is required for cleavage by the enzyme that cleaved theviral DNA. Therefore, ligation of this end of the synthetic DNA segmentwith a viral arm does not reconstitute a functional cleavage site forthe nuclease that cleaved the viral DNA. This method for eliminating acleavage site from a viral genome is exemplified in Example 4 byinsertion of a synthetic DNA segment comprising a multiple cloning siteinto a unique cleavage site of a viral genome.

To prevent inactivation of a viral genome as a result of modification,it is evident that the modification of a viral genome according to thepresent method must be made in a region of the viral genome that is notessential for virus multiplication in cell culture under the conditionsemployed for propagation of the resulting modified virus. DNA virusgenomic regions comprising sequences that are nonessential formultiplication in cell culture and otherwise suitable for modificationaccording to the present methods include sequences between genes (i.e.,intergenic regions) and sequences of genes that are not required formultiplication of the modified viral genome.

A nonessential site suitable for modifying a selected genome of aeukaryotic cytoplasmic DNA virus according to the present invention maybe identified by making a desired modification and determining whethersuch modification interferes with replication of that genome under thedesired infection conditions. More in particular, restriction enzymecleavage sites in a viral genome, including unique sites in that genome,are identified, for instance, by digestion of genomic DNA and analysisof the resulting fragments, using procedures widely known in the art.The genome may be disrupted by trial insertion of a short synthetic DNAsegment into a selected target cleavage site by the direct cloningmethod of the present invention. Recovery of a virus comprised of thetrial insert at the selected target site provides a direct indicationthat the target site is in a nonessential region of that genome.Alternatively, if no useful cleavage site exists at a particular genomictarget location, such a site may be introduced using either directmolecular cloning or conventional genome construction based on markerrescue techniques. In this case, successful recovery of a viruscomprised of the inserted cleavage site at the target location directlyindicates that the target location is in a nonessential region suitablefor modification according to the present invention.

Certain nonessential genomic regions suitable for practicing the presentinvention with poxviruses have been described. See, for instance, Goebelet al., Virology 179: 247-266 (1990), Table 1, the disclosure of whichis hereby incorporated herein by reference.

In further embodiments of the method, at least a portion of the DNAsequence which is inserted into the first viral genome is undertranscriptional control of a promoter. In certain embodiments, thispromoter is located in the DNA sequence that is inserted into the firstviral genome and, therefore, controls transcription of that portion ofthe inserted DNA sequence downstream from the promoter. This approach isexemplified by insertion into a poxviral genome of a gene cassettecomprising a promoter functionally linked to an open reading frame, asdescribed in Examples 1 through 5.

In another preferred embodiment, the promoter controlling transcriptionof the DNA sequence that is inserted into the first viral genome islocated in the modified viral genome upstream of the inserted DNAsequence. This approach is illustrated by insertion of a cDNA encodingthe human von Willebrand factor protein into a multiple cloning sitethat is functionally linked to an upstream promoter in a vaccinia virusvector, as described in Example 7.

In certain embodiments, the promoter controlling the inserted DNAsequence is recognized by an RNA polymerase encoded by the modifiedviral genome. Alternatively, this promoter might be recognized only byan RNA polymerase encoded by another genome, for example, another viralor cellular genome. For example, this RNA polymerase might be abacteriophage T7 polymerase that is encoded by another cytoplasmic DNAvirus genome or by the genome of a modified host cell. The T7 polymeraseand promoter have been used, for instance, in recombinant poxviruses toenhance expression of an inserted DNA sequence. See, for example, Fuerstet al., J. Mol. Biol. 205:333-348 (1989). Provision of the T7 RNApolymerase on a separate genome is used to prevent expression of a DNAsequence inserted into the modified poxvirus genome except when theseparate genome is present.

In still other embodiments, the promoter controlling the insert issuitable for initiation of transcription by a cytoplasmic DNA virus RNApolymerase. In some embodiments, the promoter comprises a modificationof a DNA sequence of a naturally-occurring viral promoter. One suchembodiment is exemplified by use of a “synthetic” vaccinia viruspromoter, such as the “S3A” (bases 21-107 of SEQ ID NO:13) and “S4”(bases 21-114 of SEQ ID NO:14) promoters described, inter alia, inExamples 5 and 6.

The eukaryotic cytoplasmic DNA virus genomic construction method of thepresent invention further comprises a step of introducing the modifiedDNA molecule comprising the modified viral genome into a first host cellwhich packages the modified DNA molecule into infectious modifiedcytoplasmic DNA virus virions. The modified DNA molecule is introducedinto the first host cell by a method suitable for transfection of thatfirst host cell with a DNA molecule, for instance, by methods known inthe art for transfection of other DNA's into comparable host cells. Forexample, in a preferred embodiment, the modified DNA is introduced intothe first host cell using the calcium phosphate precipitation techniqueof Graham and van der Eb, Virology 52:456-467 (1973).

In a preferred embodiment, this method for producing a modifiedeukaryotic cytoplasmic DNA virus further comprises a step of infectingthe first host cell with a second cytoplasmic DNA virus comprising asecond cytoplasmic DNA virus genome which is expressed to package themodified DNA molecule into infectious modified cytoplasmic DNA virusvirions. In the method comprising infection of the first host cell witha second virus, introducing the recombinant DNA molecule into the firsthost cell is carried out advantageously about one hour after infectingthe first host cell with the second virus.

In another embodiment of this method, the necessary packaging functionsin the first host cell are supplied by a genetic element other than acomplete genome of a second virus, such as a plasmid or other expressionvector suitable for transforming the first host cell and expressing therequired helper virus functions. Use of a nonviral genetic element toprovide helper functions enables production of genetically stable helpercells that do not produce infectious helper virus. Use of such a helpercell as a first host cell for packaging of a modified DNA moleculeadvantageously produces only virions comprised of that modified DNA.

In the method comprising infection of the first host cell with a secondvirus, the second virus is selected so that expression of the secondviral genome in the first host cell packages the modified DNA moleculeinto infectious virions comprised of the modified viral genome. Pursuantto the present invention, it is feasible to effect intracellularpackaging of a modified DNA comprising a eukaryotic cytoplasmic DNAvirus genome by transfection into cells infected with a closely relatedvirus. For instance, DNA of a first poxvirus genus is packaged by a hostcell infected with a second poxvirus of the same poxvirus subfamily,whether from the same or a different genus.

In certain embodiments, expression of the second viral genome in thefirst host cell produces infectious virions comprised of the secondviral genome as well as of the modified viral genome. This situationobtains, for instance, in the case of homolgous packaging of a firstpoxvirus DNA from one genus by a second poxvirus of the same genus.Here, although the transfected DNA theoretically could be packageddirectly, i.e., without transcription of the transfected genome,homologous packaging of the transfected DNA molecule probably involvestranscription and replication of both the transfected DNA and the DNA ofthe helper virus. This situation is illustrated, inter alia, withhomologous packaging of poxvirus DNA in Examples 1 and 2.

However, in other embodiments expression of the second viral genome inthe first host cell does not produce infectious virions comprised of thesecond viral genome. In cases involving heterologous packaging, forinstance, passive packaging alone cannot produce viable virus particlesfrom the transfected DNA. In such a case it is advantageous to select asecond (helper) virus which provides an RNA polymerase that recognizesthe transfected DNA as a template and thereby serves to initiatetranscription and, ultimately, replication of the transfected DNA. Thiscase is exemplified by the reactivation of a modified genome of anorthopoxvirus (vaccinia) vector by an avipox (fowlpox) helper virus in amammalian first host cell in which the avipox virus is unable to produceinfectious virions comprised of the avipoxvirus genome, as described inExample 3.

The use of a heterologous virus to package the modified DNA molecule,such as the use of fowlpox or ectromelia (mouse pox) virus as a helperfor vaccinia virus constructs, advantageously minimizes recombinationevents between the helper virus genome and the transfected genome whichtake place when homologous sequences of closely related viruses arepresent in one cell. See Fenner and Comben (1958); Fenner (1959).

In certain embodiments of the method for using a helper virus for DNApackaging, the step of recovering the infectious virions comprised ofthe modified viral genome comprises a step of infecting a second hostcell with infectious virions produced by the first host cell.Advantageously, the second host cell is infected under conditions suchthat expression of the second viral genome in the second host cell doesnot produce infectious virions comprised of the second virus genome. Inother words, the second host cell is infected under conditions thatselect for replication of the modified virus and against the helpervirus. This method is exemplified by a method in which the modifiedgenome is a modified vaccinia virus genome, the second genome is afowlpox virus genome, and the second host cell is a mammalian cell. Inthis method, the modified virus is plaque purified in cultures of themammalian host cell in which fowlpox virus does not produce infectiousvirions, as described in Example 3.

In another embodiment in which the second host cell is infected underconditions that select for the modified virus, the modified viral genomecomprises a functional host range gene required to produce infectiousvirions in the second host cell. The second viral genome lacks thisfunctional host range gene. This embodiment is illustrated by a methodin which the modified viral genome is a modified vaccinia virus genomecomprising a functional host range gene required to produce infectiousvaccinia virus in a human (MRC 5) cell which is used as the second hostcell, as described in Example 8.

In yet another embodiment involving selection for modified virus in asecond host cell, the modified viral genome comprises a selective markergene which the second viral genome lacks, and the step of infecting thesecond host cell is carried out under conditions that select for a viralgenome expressing the selective marker gene. For example, expression ofthe selective marker gene in the second host cell may confer on thatcell resistance to a cytotoxic drug. The drug is provided duringinfection of the second host cell at a level sufficient to select for aviral genome expressing the selective marker gene. This approach isexemplified by a method for inserting a gene for the E. coli gpt geneinto a vaccinia virus genome, as in Example 1, or a fowlpox virusgenome, as in Example 2, using in each case a homologous helper viruslacking the selective marker gene.

In still another embodiment involving selection for a modified virus ina second host cell, the modified viral genome comprises a deletion of aselective marker gene that is present in the second viral genome. Here,the step of infecting the second host cell is carried out underconditions that select against a viral genome expressing that selectivemarker gene. For example, expression of a poxvirus thymidine kinase (tk)gene in the second host cell (i.e., a thymidine kinase-negative hostcell) renders the second (helper) virus sensitive to the metabolicinhibitor, 5-bromo-deoxyuridine. Example 4 describes the use of theseinhibitors during infection of a second host cell to select for avaccinia virus vector (vdTK) in which the tk gene is deleted andreplaced by a multiple cloning site.

Another aspect of the present invention relates to a eukaryoticcytoplasmic DNA virus comprised of a modified viral genome. A modifiedgenome of a cytoplasmic DNA virus within the scope of the presentinvention comprises distinct component DNA sequences which aredistinguishable from each other, for example, by routine nucleic acidhybridization or DNA sequencing methods.

In certain embodiments, for instance, the modified viral genomecomprises a first genome of a first eukaryotic cytoplasmic DNA virus.This first genome is comprised of a cleavage site for asequence-specific endonuclease that is a unique site in the firstgenome. In this embodiment, the sequences of the modified genome thatcomprises the first viral genome are homologous to a genome of anaturally-occurring eukaryotic cytoplasmic DNA virus. Further, thesequences of this first virus are interrupted by a DNA sequence ofinterest as defined hereinabove.

To determine whether this sequence is inserted into a unique cleavagesite in the first viral genome, as required for this embodiment of amodified viral genome, the sequences immediately flanking the insert arecompared with sequences of cleavage sites for sequence-specificendonucleases.

In one form of this embodiment in which a DNA sequence is inserted intoa unique cleavage site in the first viral genome, the inserted sequencein the first viral genome is flanked by two identical intact cleavagesites for a sequence-specific endonuclease and these two sites are theonly sites for this nuclease in the complete modified genome. Each ofthese two sites is comprised of combined portions of cleaved sites fromthe first viral genome and the inserted DNA sequence.

More particularly, each strand of a double-stranded DNA comprised of acleavage site for a sequence-specific endonuclease may be considered tocomprise a complete cleavage site sequence (S_(L)S_(R)) consisting of aleft cleavage site sequence (S_(L)) and a right cleavage site sequence(S_(R)) separated by the monophosphate linkage that is disrupted bycleavage with the appropriate nuclease. In certain forms of thisembodiment, insertion of a DNA sequence into a unique restriction sitereproduces two complete sites flanking the insert.

In other forms of this embodiment, however, insertion of the DNAsequence into a unique cleavage site does not recreate the originalcleavage site at each end of the inserted DNA sequence. See, forinstance, the method for elimination of a cleavage site described inExample 6. Thus, the inserted DNA may be flanked at one end (e.g., theleft end) by a complete cleavage site (S_(L)S_(R)) while the right endterminates in a sequence that differs from S_(L) directly linked to anS_(R) sequence in the first viral genome. More generally, in anymodified viral genome of this invention, the DNA sequence inserted intoa unique site in a first viral genome will be flanked by two thematching parts (S_(L) and S_(R)) of a cleaved site which does not occurin the modified viral genome outside of the inserted DNA.

In other embodiments, the modified viral genome is comprised of a DNAsequence that is inserted between two unique sites in the first viralgenome. In this case, if the first viral genome is a naturally-occurringgenome of a eukaryotic cytoplasmic DNA virus, the insert will beencompassed by viral sequences separated from the foreign DNA sequenceat least by recognizable SL and SR portions of the two differentoriginal cleavage sites.

In additional embodiments, the modified viral genome comprises a uniquecleavage site located in a DNA sequence that is not naturally-occurringin a genome of a eukaryotic cytoplasmic DNA virus. In this case, thisforeign DNA is not separated from the natural viral DNA sequences byrecognizable S_(L) and S_(R) portions of cleavage sites. In certainforms of this embodiment, the first foreign DNA sequence is interruptedby a second foreign DNA sequence inserted into a unique cleavage site inthe first sequence or between two such sites in the first sequence. Inthese embodiments the second foreign DNA is separated from the firstforeign DNA sequences by recognizable S_(L) and S_(R) portions ofsequence-specific endonuclease cleavage sites. In this case, allsequences surrounding this second foreign DNA sequence comprise thegenome of the first virus according to this invention.

Preferred embodiments of modified eukaryotic cytoplasmic DNA viruses ofthis invention include a first major embodiment in which the modifiedviral genome comprises (I) a first genome of a first eukaryoticcytoplasmic DNA virus that is comprised of a cleavage site for asequence-specific endonuclease. This site is a unique site in the firstviral genome. The modified viral genome of this embodiment alsocomprises (II) a first DNA sequence of interest. This DNA sequence isinserted into the unique site in the first cytoplasmic DNA virus genome.

In one variation of this first embodiment of a modified eukaryoticcytoplasmic DNA virus, the first viral genome comprised of the uniquesite is a naturally-occurring viral genome. This variation isexemplified herein by a modified poxvirus genome comprised of anaturally-occurring vaccinia virus genome which has unique cleavagesites for the bacterial restriction endonucleases NotI and SmaI, asdescribed in Examples 1 and 3. In this embodiment, the first DNAsequence of interest, which is inserted into the unique site, isexemplified by an E. coli gpt gene driven by a naturally-occurringvaccinia virus promoter inserted into the NotI site (Example 1) or intothe SmaI site (Example 3) of a vaccinia virus genome.

In a second form of this first embodiment of a modified virus, the firstviral genome comprised of the unique site also comprises a second DNAsequence not naturally-occurring in a viral genome. Furthermore, thissecond DNA sequence includes the unique site for insertion of the firstDNA sequence. This variation is exemplified herein by a modified fowlpoxvirus genome comprising a DNA sequence encoding an Escherichia coliβ-galactosidase gene, as described in Example 2. This bacterial geneincludes a cleavage site for the bacterial restriction endonuclease NotIthat is unique in the modified fowlpox virus genome and, therefore, isparticularly convenient for insertion of foreign DNA sequences.

In another variation of this first embodiment of a modified virus, atleast a portion of the first DNA sequence that is inserted into theunique site is under transcriptional control of a promoter. In someinstances, the promoter is located in the first DNA sequence that isinserted into the first viral genome. This holds, for instance, when theinserted DNA comprises a gene cassette including a promoter and afunctionally linked gene, as described, inter alia, in Examples 1 and 2.

In a second embodiment of a modified cytoplasmic DNA virus of thisinvention, the modified viral genome comprises (I) a first viral genomecomprised of a first and a second cleavage site for a sequence-specificendonuclease where each of these sites is unique in the first virusgenome. In one preferred variation of this embodiment, the first viralgenome comprises a multiple cloning site comprised of several uniquecleavage sites.

In this second embodiment, the modified viral genome also comprises (II)a first DNA sequence not naturally-occurring in a genome of a eukaryoticcytoplasmic DNA virus, and this first DNA sequence is inserted into thefirst viral genome between the first and second unique cleavage sites.

In a third embodiment of a modified cytoplasmic DNA virus of thisinvention, the modified viral genome comprises (I) a first viral genomecomprised of a first DNA sequence not naturally-occurring in a genome ofa eukaryotic cytoplasmic DNA virus. This first DNA sequence is comprisedof a cleavage site for a sequence-specific endonuclease that is a uniquesite in the modified viral genome. The modified viral genome of thisembodiment further comprises (II) a promoter located such that a DNAsequence inserted into the unique site is under transcriptional controlof the promoter. This first DNA sequence does not have a translationstart codon between the promoter and the unique site used for insertionof a DNA sequence. This embodiment is exemplified by the vaccinia virusvector (vS4) described in Example 6, which has a “synthetic” poxviruspromoter located such that this promoter controls transcription of a DNAsequence inserted into a multiple cloning site designed for insertion ofopen reading frames.

Another aspect of the present invention relates to a DNA moleculecomprising a modified viral genome of a modified eukaryotic cytoplasmicDNA virus of this invention. In a preferred embodiment, this DNAmolecule is prepared by extraction of genomic DNA molecules from virionsof a modified eukaryotic cytoplasmic DNA virus of this invention, orfrom cells infected with a modified virus of this invention. Methodssuitable for extracting modified viral genomic DNA's from virions areknown in the art. In addition, suitable methods for preparing DNA ofeukaryotic cytoplasmic DNA viruses are described herein in Example 1.

Still another aspect of the present invention relates to genomic DNAarms of a eukaryotic cytoplasmic DNA virus of this invention. Thesegenomic DNA arms are useful for direct molecular cloning of viralgenomes comprising foreign DNA's. More particularly, this aspect of theinvention relates to two DNA molecules, the left and right genomic armsof a modified viral genome of a eukaryotic cytoplasmic DNA virus. In thepractice of the direct cloning method of this invention, describedabove, either one or both of these arms may consist entirely of a DNAsequence that is naturally-occurring in a cytoplasmic DNA virus. But thenovel DNA molecule of the present aspect of this invention is a modifiedarm of a viral genome, in other words, a DNA molecule comprising one endof a modified viral genome of a eukaryotic cytoplasmic DNA virus. Thisend of the modified viral genome comprises a DNA sequence of interestwhich distinguishes this DNA molecule from genomic arms consisting ofonly a sequence that is naturally-occurring in a cytoplasmic DNA virus.In addition, the modified viral genome from which the novel arm derivesis comprised of a unique cleavage site for a sequence-specificendonuclease. Furthermore, this DNA molecule has a terminus that ishomologous to a product of cleaving the unique site in the modifiedviral genome with the sequence-specific endonuclease.

In a preferred embodiment, this DNA molecule comprising a genomic arm isproduced by cleavage of genomic DNA of a modified virus at a unique sitefor a sequence-specific endonuclease. Alternatively, this DNA moleculemay be produced by modifying another DNA molecule to produce a terminusthat is homologous to a terminus produced by cleaving a unique site in amodified viral genome. For instance, a DNA molecule according to thisaspect of the invention may be produced from an arm of anaturally-occurring genomic viral DNA. The required DNA molecule may beproduced from such a naturally-occurring viral arm, for example, byligation to a synthetic “adaptor” DNA segment comprised of a cohesiveend derived from cleavage site that is not present in the first viralgenome. In this instance the end of the first viral genome and theligated adaptor together comprise one end of a modified viral genome.Accordingly, this particular DNA molecule is not produced by cleavage ofa modified viral genomic DNA, but it does comprise a terminus that ishomologous to a terminus that is produced by cleaving a unique site in amodified viral genome.

In another embodiment of a modified viral DNA arm of the presentinvention, the DNA sequence not naturally-occurring in a genome of aeukaryotic cytoplasmic DNA virus is comprised of the cleavage site for asequence-specific endonuclease that is unique in the modified viralgenome. This cleavage site further comprises a left cleavage sitesequence (S_(L)) for the left genomic arm, or the right cleavage sitesequence (S_(R)) for the right genomic DNA arm, occurring completecleavage site sequence (S_(L)S_(R)) being unique in the modified viralgenome. This embodiment is exemplified, inter alia, by DNA arms producedfrom a fowlpox virus vector by the bacterial restriction endonucleaseNotI, as described in Example 2, or by arms of a vaccinia virus vector(vS4) cleaved at any of several unique sites of an inserted multiplecloning site, as described in Example 6.

Yet another aspect of the present invention relates to a kit for directmolecular cloning of a modified viral genome of a eukaryotic cytoplasmicDNA virus. This kit comprises (I) purified DNA molecules of thisinvention. These DNA molecules comprise either genomic viral DNA arms ofthis invention or a complete, intact modified viral genome of thisinvention, or both. The viral DNA arms are useful for direct ligation toforeign DNA segments to be cloned, while the intact viral DNA's areuseful for cloning after cleavage, for instance, with asequence-specific endonuclease at a site that is unique in the modifiedviral genome.

The kit further comprises (II) a DNA ligase and (III) solutions of abuffer and other reagents suitable for ligation of DNA segments togetherto produce a modified DNA molecule comprising said modified viralgenome. A suitable buffer and reagents for ligation are described, forinstance, in Example 1.

In one embodiment, this kit further comprises a plasmid comprised of agene expression cassette flanked by sites for cleavage with asequence-specific endonuclease. When cleaved by the appropriatesequence-specific endonuclease, the sites flanking the cassette produceends that are compatible for insertion of this cassette into a uniquecleavage site of the modified viral genome that is encoded by the DNAmolecule.

In another embodiment, the cloning kit further comprises a first hostcell and a second (helper) virus suitable for packaging the modifiedviral genome into infectious virions.

Yet another aspect of the present invention relates to plasmids whichare particularly suited to serve as intermediates in the construction ofmodified cytoplasmic DNA virus vectors of this invention.

According to one embodiment of this aspect, there is provided a plasmidcomprising a DNA segment having at each end the same cleavage site for asequence-specific endonuclease. This site is also a unique site in afirst cytoplasmic DNA virus genome according to the present invention.This DNA segment comprises a multiple cloning site comprised of severalclosely adjacent sequence-specific endonuclease cleavage sites that areunique in the plasmid and, therefore, useful for insertion of foreignDNA segments into the plasmid.

This plasmid is useful for insertion of genes into a unique cleavagesite of the DNA segment for subsequent transfer of that segment into aunique cleavage site of a cytoplasmic DNA virus using the directmolecular cloning method of this invention. This plasmid is exemplifiedby the plasmid pN2 (see Example 1, FIG. 1.3) which has a DNA segmentcomprising a multiple cloning site flanked by NotI sites and containingthe following additional bacterial restriction enzyme cleavage sites inthe stated order: XbaI, SpeI, BamHI, SmaI, PstI, EcoRI, EcoRV, HindIIIand ClaI.

Another plasmid of the present invention comprises a DNA segment havingat each end a cleavage site that is a unique site in a cytoplasmic DNAvirus. The DNA segment of this plasmid also comprises severalrestriction enzyme cleavage sites that are unique in the plasmid. ThisDNA segment further comprises a selective marker gene (e.g., an E. coligpt gene) under transcriptional control of a cytoplasmic DNA viruspromoter (e.g., the vaccinia virus P7.5 promoter). This plasmid isexemplified by two plasmids designated pN2-gpta and pN2-gptb whichcontain a DNA segment flanked by NotI sites and comprising an E. coligpt gene under transcriptional control of a vaccinia virus P7.5promoter. This plasmid was created by insertion of the promoter-genecassette into the SmaI site of the plasmid pN2, as described in FIG.1.3.

In a further modification of the above plasmid, the DNA segment furthercomprises a second poxvirus promoter operatively linked to a DNAsequence comprising a restriction endonuclease cleavage site. Thisplasmid, as exemplified by the plasmid pN2gpt-S3A (FIG. 4.6) can be usedto insert open reading frames lacking their own initiation codon fortransfer into a vaccinia virus vector. Similarly, the plasmid pN2gpt-S4(FIG. 4.6) can be used to insert complete open reading frames includingan AUG translation start codon.

In another embodiment, this plasmid further comprises a DNA sequenceencoding human plasminogen, wherein the DNA sequence is operativelylinked to the poxvirus promoter and start codon. This plasmid isexemplified by plasmid pN2gpt-GPg, encoding human glu-plasminogen, andby plasmid pN2gpt-LPg, encoding lys-plasminogen, in which the codingregion for amino acids 1-77 of human plasminogen is deleted (FIGS. 5.2and 5.3).

In a related form, this plasmid further comprises a DNA sequenceencoding HIV-1 gp160, wherein the DNA sequence is operatively linked tothe poxvirus promoter and start codon. This is exemplified by plasmidpN2gpt-gp160, having the gp160 gene controlled by the synthetic vacciniavirus promoter S4 (FIG. 5.4).

Another plasmid of the present invention comprises a segment of acytoplasmic DNA virus genome in which the viral tk gene is located. Inthis plasmid, the coding region of the tk gene has been modified(deleted) to prevent expression of active tk enzyme. This plasmid isuseful as an intermediate in construction of a cytoplasmic DNA virusvector having a defective tk gene, using conventional methods of markerrescue, as described for the vaccinia virus tk gene, using plasmidpHindJ-3. In a related embodiment, a plasmid comprising a modified tkgene region of a cytoplasmic DNA virus further comprises a multiplecloning site comprised of several closely adjacent sequence-specificendonuclease cleavage sites that are unique in the plasmid. Furthermore,each of these sites is absent in a cytoplasmic DNA virus into which themodified tk gene region is to be inserted. Therefore, after insertion ofthe modified tk gene region comprising these unique sites into thatviral genome, these sites are useful for insertion of foreign DNAsegments into the cytoplasmic DNA virus genome carrying the modified tkgene region, according to the direct cloning method of the presentinvention.

This plasmid comprising a modified tk gene region containing a multiplecloning site is exemplified by plasmid pHindJ-3 in which the modifiedvaccinia virus tk gene region of plasmid pHindJ-2 has inserted amultiple cloning site with the unique sites NotI, SmaI, ApaI and RsrII,flanked by SfiI sites (FIG. 4.2). To further facilitate forced cloningin a vaccinia virus vector, each of the two SfiI sites is also madeunique in the vector by exploiting the variable nature of the SfiIrecognition sequence, as detailed in Example 4.

In still another embodiment, a plasmid comprises a sequence-specificendonuclease cleavage site that is unique in the genome of that virus.Such plasmids are particularly suitable for construction of geneexpressions cassettes for transfer into a vector having theaforementioned unique site. The plasmid pA0 exemplifies the basicplasmid that contains a master cloning site comprised of the uniquesites of the master cloning site of the vdTK vaccinia virus vector (FIG.4.3). The related plasmids pA1 and pA2 were designed for insertion ofDNA segments, for instance, synthetic or natural promoter fragments andwere constructed by inserting into the Xhol site of pA0 a linkercomprising a second multiple cloning site of frequently cutting enzymesthat do not cleave pA0. Both plasmids have the same structure except forthe orientation of the second multiple cloning site (FIG. 4.3).

In yet another embodiment, a plasmid comprises a poxvirus promoteroperatively linked to a translational start codon, wherein this startcodon is immediately followed by a second restriction endonucleasecleavage site suitably arranged to permit translation of an open readingframe inserted into the second restriction endonuclease cleavage site.This plasmid is exemplified by plasmids pA1-S1 and pA2-S1 (comprisingSEQ ID NO:10) which provide the strong synthetic poxvirus promoter S1(bases 21-194 of SEQ ID NO:9), including a translational start codon,followed by a single EcoRI site suitable for insertion of open readingframes that do not have an associated start codon (FIGS. 4.4A and 4.4B).Plasmids pA1-S2 and pA2-S2 are similar to pA1-S1 and pA2-S1 but have adifferent poxvirus promoter, S2 (FIGS. 4.5A and 4.5B, bases 21-73 of SEQID NO:11).

In a related embodiment, the plasmid above further comprises a DNAsequence encoding human prothrombin, wherein said DNA sequence isoperatively linked to said poxvirus promoter and said start codon. Thisplasmid is exemplified by the plasmid pA1S1-PT (FIG. 5.1) in which amodified prothrombin cDNA is inserted into the single EcoRI site of theplasmid pA1-S1.

Another plasmid of the present invention comprises a modified EcoRI Kfragment of vaccinia virus DNA from which the K1L host range gene isdeleted. The helper virus vdhr lacking both the K1L and C7L host rangegenes is constructed from the C7L-negative strain WR-6/2 by markerrescue with a modified EcoRI K fragment from which the K1L host rangegene is deleted. See FIG. 8.1. This modified EcoRI K fragment comprisesa selective marker gene (the E. coli gpt gene) to facilitate selectionfor recombinant WR-6/2 genomes comprising the modified EcoRI K fragmentusing intracellular marker rescue as described by Sam and Dumbell(1981). The exemplifying plasmid is designated pEcoK-dhr (FIG. 8.1).

In a further step pEcoK-dhr is linearized with NotI and ligated with a1.1 kb P7.5-gpt gene cassette derived from plasmid pN2-gpta (Example 4)by NotI digestion. The resulting plasmid pdhr-gpt (FIG. 8.1) is used inmarker rescue experiments to generate the helper virus vdhr according tothe marker rescue method of Sam and Dumbell (1981).

The present invention is further described below with regard to thefollowing illustrative examples. Certain constructs are illustrated withtables detailing their characteristics. In those tables, the followingabbreviations are used:

CDS=coding sequence

rc=reverse complementary sequence

rcCDS=reverse complementary coding sequence; arabic numbers arepositions of nucleotides

ATG=translational start codon

EMBL ID=Identifier in EMBL DATABANK

EXAMPLE 1 Direct molecular cloning of foreign DNA comprising a selectivemarker gene (the gpt gene of E. coli) into a unique (NotI) cleavage sitein the genome of an orthopoxvirus (vaccinia)

This example demonstrates direct molecular cloning of a gene expressioncassette into a poxvirus genome, according to the present invention, byintracellular packaging of genetically engineered poxvirus DNA. Inaddition, this example illustrates use of a genetic selection procedurefor efficient recovery of modified vaccinia viruses containing aninserted selective marker gene. The experimental results also revealthat recombination frequently occurs between the DNA to be packaged andthat of the infecting helper virus during packaging when the helpervirus DNA is homologous with the DNA to be packaged.

More particularly, a first direct molecular cloning experiment describedbelow shows that a marker gene (gpt-gene) cassette can be inserted as aNotI restriction fragment in NotI-cleaved vaccinia virus DNA andsubsequently packaged in vaccinia virus-infected mammalian cells. One ofnine plaques examined comprised virus having the predicted structure fora single insert of the gpt-gene in the “a” orientation (see FIG. 1.11).The structure of this clone (designated vp7) was stable during largescale replication in the absence of the selection agent.

In a second series of cloning experiments, seven of twelve clonesexamined had the expected structure. In this series, however, four smallplaques (E1-E4) of slowly replicating viruses were included, althoughpreferably these are not normally selected in the practice of thepresent invention. Recombinants having multiple inserts of the selectivemarker gene were also obtained under selective conditions. The stabilityof these multiple inserts was not examined in the absence of theselective agent which is known to stabilize certain otherwise unstablestructures. See Falkner and Moss, J. Virol. 64:3108-3111 (1990).

The relatively low yield of predicted structures is not expected giventhe known precision of genetic engineering methods for site-specificcleavage and ligation of DNA molecules. However, the particular sequenceselected for insertion in this model system, the gpt-gene cassette,comprised vaccinia virus DNA sequences of the P7.5 promoter which arehomologous to two endogenous promoters in the vaccinia vector whichdrive two vaccinia virus 7.5-kD polypeptide genes located within theinverted terminal repetitions of the vaccinia genome. See Venkatesan etal., Cell 25:805-813 (1981). This P7.5 promoter has been used toconstruct vaccinia virus recombinants by conventional intracellularrecombination and can be stably integrated into the vaccinia thymidinekinase gene. Mackett and Smith (1986). Occasionally, however, submolaramounts of DNA fragments appear during analyses of conventionalrecombinants, which may result from secondary recombination events.Where a P7.5 promoter is inserted near the endogenous P7.5 promoters(i.e., within several kilobases), only recombinants that have aninverted repeat structure are stable, and this observation has beenexploited to develop a deletion procedure based on insertion of atandemly repeated P7.5 promoter segment. Spehner et al., J. Virol.64:527-533 (1990).

In the present case of insertion of the gpt-gene cassette into the NotIsite of vaccinia virus, the distance between the P7.5 promoters of theleft inverted terminal repetition and that of the inserted cassette isabout 30 kb, probably close enough to cause destabilizing secondaryrecombination events. In fact, only the structures of a few slowlyreplicating, unstable clones had an insert in the “b” orientation whichwould produce a tandem repeat arrangement of the inserted and endogenouspromoters. Thus, the rare occurrence of this structure can be explainedmost likely by the closeness of the locations of the P7.5 promoters ofthe gpt-gene cassette and the endogenous P7.5 promoters and the knowninstability of tandemly repeated copies of the P7.5 promoter.

In contrast, the virus vp7 and several other isolates (A1, A4, C1 andC2) had inserts in the “a” orientation and were stable. The structuralanalysis of one isolate, C4, was consistent with a head-to-tail doubleinsert.

The titers of packaged gpt-gene positive viruses in the second series ofcloning experiments (five different samples) were approximately 1×10⁵pfu per 8×10⁶ cells, while in the first experiment a titer of 1-2×10²pfu was obtained from the same number of cells. The titer of modifiedviruses will be influenced by several factors, including ligation andpackaging efficiencies, reaction and culture conditions in the cloningprocedure, and by the amount of care taken to avoid shearing of the highmolecular vector DNA during handling. Titers of about 10⁵ plaque formingunits (pfu) per 8×10⁶ cells are generally expected under the standardconditions described hereinbelow.

While the present example shows that the unique intergenic NotI site ofvaccinia virus can be used for insertion of foreign DNA, it alsoillustrates the need to consider whether a proposed insert may containviral sequences of a type and orientation that are known or likely tocause instability of modified viruses. Inserts lacking homology withviral sequences near the insertion site (e.g., within 30 kb) are to bepreferred for stability. Accordingly, inserts comprising only shortsynthetic promoter sequences that are recognized by the transcriptionsystem of the vector are preferred to those containing large segments ofviral DNA including natural promoters of the viral vector. See, forinstance, the S1 promoter in Example 4, below.

The following materials and methods were used throughout this and allsubsequent examples, except where otherwise specified.

Purification of orthopox virus and DNA: Vaccinia virus (wildtype WesternReserve (WR) strain; American Type Culture Collection No. VR 119) waspurified by two successive sucrose gradients according to Mackett et al.in DNA CLONING: A PRACTICAL APPROACH, ed. D. Glover, IRL Press (1985) p.191-211. Viral DNA was prepared by the proteinase K-SDS procedureaccording to Gross-Bellard et al., Eur. J. Biochem. 36:32-38 (1973).

Engineering of isolated poxvirus DNA: Viral DNA (typically 2 to 5 μg)was cleaved with appropriate amounts of one or more sequence-specificendonucleases (for example, the bacterial restriction endonucleaseNotI), optionally treated with calf intestine alkaline phosphatase(Boehringer, Inc.), and purified by phenol extraction and ethanolprecipitation, according to routine recombinant DNA methods. Theresulting viral DNA arms were ligated with a five to fifty-fold molarexcess of the DNA fragment to be inserted, having ends compatible forligation with the viral arms. An aliquot of the ligation reaction wasanalyzed by field inversion gel electrophoresis.

More particularly, in the second series of experiments (A-E) describedbelow, 2 μg of NotI-digested vaccinia DNA that was not treated withphosphatase were ligated with 200-600 ng of gpt-gene cassette insert ina volume of 30 μl with 5-15 units of T4 ligase for 48 h at 12° C., assummarized in Table 1.

In vivo packaging in mammalian cells: 8×10⁶ African Green monkey (CV-1)cells were infected with helper virus (either vaccinia WR wildtype orWR6/2 virus, or other viruses as indicated) at 0.2 pfu/cell for 2 h. Forthe initial demonstration of packaging with intact DNA isolated fromvirions, 20 μg of viral (vPgD) DNA were used. For packaging ofextracellularly engineered genomes, 1 μg of DNA purified from a ligationreaction were used. DNA's were transfected into cells by the calciumphosphate precipitation technique. Graham and van der Eb (1973). Thecells were incubated for 15 min at room temperature and then nine ml ofmedium (DMEM, 10% fetal calf serum, glutamine and antibiotics) per oneml precipitate were added to the cells. After four hours the medium waschanged and further incubated for two days.

Crude virus stocks were prepared according to standard procedures.Mackett et al., 1985. Plaque assays and selection conditions for the E.coli gpt gene are known in the art. See Falkner and Moss, J. Virol.62:1849-1854 (1988); Boyle and Coupar, Gene 65:123-128 (1988).

Field inversion gel electrophoresis (FIGE). Viral DNA was separated on a1% agarose gel in Tris/Acetate/EDTA buffer (40 mM Tris/20 mM glacialacetic acid/2 mM EDTA, pH 8.0) with a microcomputer controlled powersupply (Consort Model E790). To separate the whole range of fragments,four programs were run successively, as follows: program 1-5 h at 7 V/cmforward pulse (F) 6 sec, reverse pulse (R) 3 sec, pause 1 sec; program2-5 h at 7 V/cm, F 4 sec, R 2 sec, pause 1 sec; program 3-5 h at 7 V/cm,F 2 sec, R 1 sec, pause 1 sec; and program 4-5 to 10 h at 7 V/cm, F 8sec, R 4 sec, pause 1 sec.

Construction of plasmid pN2: The plasmid Bluescript II SK⁻ (Stratagene,Inc.) was digested with HindII and ligated to NotI linkers (Pharmacia,Inc.). The resulting plasmid, pN2, has a multiple cloning site flankedby NotI sites.

More particularly, the multiple cloning site of pN2 consists of thefollowing sites in the stated order: NotI, XbaI, SpeI, BamHI, SmaI,PstI, EcoRI, EcoRV, HindIII, ClaI and NotI. The inserted NotI linkersequence of pN2 and twenty bases of the 5′ and 3′ flanking regions ofpBluescript II SK- (Stratagene, Inc. La Jolla, USA) are shown in SEQ IDNO:1. The insert sequence starts at position 21 and ends at position 28.The first “T” residue at the 5′-end corresponds to position number 2266,the last “G” residue at the 3′-end to position number 2313 of theplasmid pN2.

Construction of plasmids pN2-gpta and pN2-gptb: The 1.1 kb HpaI-DraIfragment (containing the P7.5 promoter-gpt gene cassette) was isolatedfrom the plasmid pTKgpt-F1s and inserted into the SmaI site of theplasmid pN2 (FIG. 1.3). Falkner and Moss, 1988. The two resultingplasmids are orientational isomers and were designated pN2-gpta andpN2-gptb. The vaccinia virus P7.5 promoter-E. coli gpt-gene cassette andtwenty bases of the 5′-and 3′-flanking regions of pN2 are shown forpN2-gpta in SEQ ID NO:2. The insert starts at position 21 and ends atposition 1113. The A-residue of the translational initiation codon ofthe gpt gene corresponds to position 519. The T-residue of thetranslational stop codon of the gpt gene corresponds to position number975. (The first “C” residue at the 5′-end corresponds to the positionnumber 2227, the last “T” residue at the 3′-end to position number 3359of the plasmid pN2-gpta).

The reverse complementary form of the vaccinia virus P7.5 promoter-E.coli gpt gene cassette and twenty bases of the 5′- and 3′-flankingregions of pN2 are shown for pN2-gptb in SEQ ID NO:3. The insert startsat position 21 and ends at position 1113. The T-residue of the (reversecomplement of the) translational initiation codon CAT corresponds toposition 615. The A-residue of the (reverse complement of the)translational stop codon of the gpt gene corresponds to the positionnumber 159.

Other standard techniques of recombinant DNA analysis (Southern blotanalysis, PAGE, nick translation, for example) were performed asdescribed. See Sambrook et al., MOLECULAR CLONING, Cold Spring HarborLaboratory Press (1989).

Packaging of naked viral DNA: To establish conditions needed forpackaging of naked poxvirus DNA by a helper virus, intact DNA isolatedfrom virions of an exemplary recombinant vaccinia virus (vPgD) wastransfected into monkey (CV-1) cells infected with a helper virus(vaccinia WR wildtype). The selected recombinant virus has severalreadily assayable phenotypic markers, Thus, the vPgD genome hasincorporated into the viral tk locus a gene for a drug resistance marker(a gene for the enzyme xanthine-guanine-phosphoribosyl-transferase ofEscherichia coli, i.e., the “gpt” gene) and a gene for a convenientlydetected marker protein (human plasminogen). This virus was originallyconstructed from the vaccinia virus strain WR 6/2, described by Moss etal., J. Virol. 40:387-95 (1960), which has a deletion of about 9 kb and,consequently, does not express the viral major secreted 35K proteingene, as described by Kotwal et al., Nature 335:176-178 (1988). Theexpected phenotype of the packaged virus, therefore, includes:tk-negative (i.e., replication in the presence of bromodeoxy-uridine),gpt-positive (i.e., replication in the presence of mycophenolic acid andxanthine), positive for the expression of the human plasminogen gene andnegative for the expression of the secreted 35K protein.

Eight gpt-positive plaques from the above packaging experiment wereanalyzed. All were tk-negative, and, as shown in FIG. 1.1, all expressedplasminogen. Six of these isolates (lanes 5, 6, 7, 11, 12 and 14) didnot express the 35K secreted vaccinia protein and thus showed all thecharacteristics of the transfected genomic DNA. Two of the plaques alsoexpressed the 35K protein marker (lanes 4 and 13) and therefore wererecombinants between the helper wild-type virus (lanes 8 and 15) and theinput viral genomes.

This experiment established that naked poxvirus DNA extracted fromvirions is packaged when transfected into helper virus-infected cellsunder the tested conditions. Therefore, these conditions were employedfor transfection of genomic poxvirus DNA that had been modified bydirect molecular cloning, as outlined in FIG. 1.2.

Packaging of extracellularly engineered poxvirus DNA: The genome ofvaccinia virus contains a single cleavage site for the NotIsequence-specific endonuclease in the region known as the HindIII Ffragment. Inspection of the sequence around this site, identified byGoebel et al. (1990), revealed that it is located in an intergenicregion that is unlikely to be essential for viral replication. A markergene expression cassette was constructed in two plasmids (pN2-gpta andpN2-gptb; FIG. 1.3) by insertion of the E. coli gpt gene in each of thetwo possible orientations. The gpt gene was controlled by the promoterof the vaccinia virus gene coding for the 7.5 kDa protein described inCochran et al., J. Virol. 54:30-37 (1985) (labeled P1 in FIG. 1.2 andP7.5 in FIG. 1.3). The entire marker gene cassette resided on a single1.1 kb NotI fragment of these plasmids. This restriction fragment frompN2-gpta was ligated with NotI digested WR wildtype DNA and transfectedinto cells that had been infected with helper virus (WR).

In a first cloning experiment, Southern blot analyses of the genomicstructures of phenotypically gpt-positive progeny plaques was carriedout. The viral isolates were plaque-purified three times and amplifiedunder gpt-selection. The HindIII-digested DNA fragments of cells (CV-1)infected with the different viruses were separated on a 1% agarose gelby a combination of normal electrophoresis and field inversion gelelectrophoresis. The gel was then blotted and hybridized with³²P-labelled vaccinia WR DNA and a labelled probe containing gptsequences. The results confirmed that all phenotypically marker-positiveclones contained the 1.1 kb gpt insert.

FIG. 1.4 shows blots of HindIII DNA fragments from cells infected withthe nine virus isolates (lanes 4-12); plaques 2.1.1 to 7.1.1 and 10.1.1to 12.1.1). The expected 0.8 kb HindIII fragment that contains the gptsequences can be observed. In lanes 2 and 3, where HindIII-digestedwild-type virus DNA (100 and 50 ng, respectively) were loaded, nocross-hybridization to viral sequences was visible.

In the next experiment, total DNA's of CV-1 cell cultures infected withthe nine different plaques were digested with NotI. The Southern blotanalysis of the separated fragments is shown in FIG. 1.5. Unexpectedly,two bands were visible in most virus isolates, the predicted 1.1 kbinsert and a second, larger fragment. Only plaque number 7.1.1 (lane 8)showed the expected single 1.1 kb band. While the hybridization signalof the larger fragment is equally strong in all examined DNA's, theintensity of the 1.1 kb band varied from DNA to DNA, indicating that the1.1 kb insert may be present in different molar amounts in differentgenomes. The wildtype virus control (lane 2) did not hybridize to thegpt-gene probe.

The same blot was also hybridized with a vaccinia virus DNA probe. Threefragments are expected, of about 145 kb, 45 kb and 1.1 kb. The blotpatterns obtained included the expected bands but also showed anadditional band at about 5 kb. Only plaque 7.1.1 did not have theunexpected 5 kb band.

The orientation of the DNA insert in selected engineered vacciniagenomes was also investigated by Southern blot analysis. As shown inFIG. 1.2, the insert in viral DNA's may be in either the “a” or “b”orientations which are distinguishable by digestion of the DNA's withappropriate restriction enzymes. Following preliminary analyses, isolate7.1.1 was designated clone vp7, appeared to have the genomic structureof the expected modified virus and therefore was expanded and purified.The DNA of this clone was compared with that of wildtype virus bydigestion with several restriction enzymes and separation on an agarosegel by field inversion gel electrophoresis (FIG. 1.6). In a NotI digestof vp7 stained with ethidium bromide (lane 2), only the 145 kb and 45 kbbands contained sufficient DNA mass to be visible, since the band forthe 1.1 kb insert was estimated to contain only about 3 ng DNA. However,hybridization with a gpt-specific probe revealed a weak band at 1.1 kb(FIG. 1.7, lane 2). In digests with HindIII, the expected bands at 1.4and 0.8 kb were observed. As predicted, the 0.8 kb band hybridized withthe gpt-gene probe (FIGS. 1.6 and 1.7, lanes 4). In double digests withNotI and HindIII, the expected 0.8 kb fragment was also observed (FIGS.1.6 and 1.7, lanes 6).

In digests of vp7 DNA with PstI, a predicted 4.1 kb fragment containinggpt sequences was observed (FIGS. 1.6 and 1.7, lanes 8; the 4.1 kbethidium bromide-stained band in FIG. 1.6 is actually a doublet of 4.1kb fragments, one of which contains the gpt insert). Upon cleavage withboth PstI and NotI, the gpt gene cassette was released as a 1.1 kbfragment (FIGS. 1.6 and 1.7, lanes 10).

The patterns of digests obtained with these and other restrictionnucleases, including SalI (FIGS. 1.6 and 1.7, lanes 12), are consistentwith the interpretation that vp7 is a stable modified virus that has thegpt-gene integrated into the NotI site of the vaccinia virus genome inthe “a” orientation (see FIG. 1.11).

A second series of cloning experiments were done under slightly modifiedconditions (see Table 1 and methods, above). Five different ligationreactions (A-E) were set up containing constant amounts of NotI-cleavedvaccinia vector DNA and increasing amounts of insert DNA. Packaging wasdone under standard conditions in vaccinia virus-infected CV-1 cells.The titers of gpt-positive vaccinia viruses in all cases were about1×10⁵ pfu per 8×10⁶ cells. The plaque population in all cloningexperiments was heterogeneous in size: about half had a normal sizewhile the other half were smaller than normal.

TABLE 1 Effect of ratio of insert to vector DNA on yield of modifiedviruses Experiment A B C D E NotI-cleaved 2 2 2 2 2 vector DNA (μg) 0.20.2 0.4 0.4 0.6 gpt-gene insert (μg) insert molar excess 17 17 34 34 51T4 ligase (units) 5 15 5 15 15 gpt-positive virus (10⁵) 1.12 0.88 0.960.96 1.16 (pfu/8 × 10⁶ cells)

Twelve gpt-positive plaques were isolated, four each in three seriesdesignated series A, C and E, comprising 8 normal-sized (large) plaques(A1-4 and C1-4) and 4 small plaques (E1-4). Each of these plaques wasanalyzed by infecting CV-1 cells in gpt-selective medium, isolatingtotal cell DNA's and digesting them with restriction nucleases,separating the fragments by FIGE and blotting the onto a nitrocellulosemembrane.

In FIG. 1.8, the NotI-digested DNA samples hybridized with the vacciniavirus DNA probe are shown (A1-4, lanes 1-4; C1-4, lanes 5-8; E1-4, lanes9-12). Due to overloading of the gel, the bands smeared somewhat but theessential features are clearly visible. The 145 kb and the 45 kb bandsprovided the main signal. A weak band at about 5 kb of unknown origincan be seen in some of the samples. The 1.1 kb band, comprising theP7.5-promoter-gpt gene cassette, makes up only 0.6% of the viral genomeand contains only 300 bp of hybridizing sequence (i.e., the P7.5promoter). Therefore, this band was not expected to give a detectablehybridization signal under the conditions used. In a longer exposure ofthe blot, when the larger bands are heavily overexposed, the 1.1 kbbands did become visible.

As to the nature of the small plaque phenotype, small plaques E1, E3 andE4 produced only weak hybridization signals (FIG. 1.8, lanes 9-12)indicating that the virus in these plaques had not replicated asextensively as those in normal-sized plaques (lanes 1-8), while isolateE2 failed to produce a detectable amount of DNA (lane 10).

The samples shown in FIG. 1.8 were also hybridized with the gpt geneprobe (FIG. 1.9). The expected single hybridization signal was obtainedwith plaques A1, A4, C1, C2, C4, E3 and E4 (FIG. 1.9, lanes 1, 4, 5, 6,8, 11 and 12). The plaque A2 (lane 2) had the gpt gene integrated intothe 45 kb band. (The weak signal in the 145 kb band may be due tocontamination with a second minor species or to secondary recombinationevents.) The plaque A3 (lane 3) has gpt gene sequences integrated intothe 145 kb and 45 kb bands, while the plaque C3 (lane 7) has anintegration of those sequences into the 145 kb band and into the NotIsite. The plaques A2, A3 and C3 are probably recombinants that arose byillegitimate intracellular recombination of homologous sequences presentin the model gene cassette insert and in the inverted repetitions of theviral DNA.

As with the vaccinia virus DNA probe, the small plaques E1-E4 producedonly weak hybridization signals (FIG. 1.9, lanes 9-12) indicating thatthe virus in these plaques had not replicated as extensively as those innormal-sized plaques. The wildtype virus DNA and uninfected CV-1 cellDNA did not hybridize with the gpt gene probe (FIG. 1.9, lanes 13 and15).

The orientation and copy number of the gpt gene inserts were determinedby digesting the samples shown in FIG. 1.9 with PstI and Southern blotanalysis. The expected sizes of new PstI fragments resulting frominsertion of the gpt gene are shown in FIG. 1.11. Hybridization with thegpt gene probe revealed that the patterns of plaques A1, A4, C1 and C2(FIG. 1.10 lanes 1, 4, 5 and 6) comprised a single PstI fragment of 4.1kb as expected for a single insert in the “a” orientation (FIG. 1.11).For plaque El, a weak hybridization signal from a 21 kb band, which wasobserved only in long exposures of the blot, was consistent with the “b”orientation of the gpt gene insert.

The structures of the viral DNA's from plaques C4 and E3 (FIG. 1.10,lanes 8 and 11) were consistent with double tandem inserts in the “b”orientation. In this case hybridizing fragments of 21 kb and 1.1 kb areexpected (FIG. 1.11). The structure of the virus in plaque E4,comprising two fragments of 4.1 kb and 1.1 kb, is consistent with atandem insertion of two gpt genes in the “a” orientation. The DNA fromplaques A2, A3 and C3 exhibited more complex patterns indicative ofinsertions at multiple sites which were not further analyzed.

In summary, in the second cloning experiment five of eight normal-sizedplaques had genomic structures expected for insertion of a single gptgene cassette into the unique NotI site of the vaccinia virus genome.The slower growing small-sized plaques exhibited unstable structureswhich were lost during subsequent plaque purification steps.

EXAMPLE 2 Direct molecular cloning of a selective marker gene (E. coligpt) into a unique (NotI) cleavage site of a modified avipoxvirus genome(fowlpox virus clone f-TK2a)

This example illustrates the general applicability of direct molecularcloning of modified cytoplasmic DNA virus genomes by illustrating anapplication to modified avipoxvirus genomes that are engineered in vitroand packaged in vivo. Avipoxviruses have the largest genomes of thepoxvirus family. The genome of fowlpox virus (FPV) is about 300 kb insize, and heretofore FPV recombinants expressing foreign genes have beenconstructed only by marker rescue techniques. See, for example, Boyleand Coupar, Virus Res. 120:343-356 (1988); Taylor et al., Vaccine5:497-503 (1988).

The present example illustrates production of a modified fowlpox virusby direct molecular cloning of a gene expression cassette consisting ofa poxvirus promoter driving the E. coli gpt gene into a unique NotI sitein the genome of a recombinant fowlpox virus, f-TK2a. This NotI site islocated in a lacZ gene which was previously inserted into thisrecombinant by intracellular recombination. Engineered DNA is packagedin primary chicken embryo fibroblasts infected with the HP2 helperfowlpox virus which replicates more slowly than the f-TK2a recombinant.Selection for gpt-positive plaques leads to isolation of engineeredfowlpox viruses. Since the lacZ marker gene is inactivated by aninsertion at the NotI site, the progeny virus are distinguished fromvector virus lacking an insert, by a colorless phenotype in the blueplaque assay for lacZ gene expression.

Purification of fowlpox virus and DNA: The fowlpox virus (FPV) strainHP1 and the attenuated strain HP1.441 (passage number 441 of HP1) wereobtained from A. Mayr, Munich. Mayr and Malicki, Zentralblatt f.Veterinärmedizin, Reihe B, 13:1-12 (1966). The fowlpox virus strain HP2was derived from HP1.441 by plaque purification. Primary chicken embryofibroblasts (CEF) were prepared as described in European PatentApplication No. 0 338 807. The cells were grown in tissue culture medium199 (TCM 199; Gibco BRL) supplemented with 5% fetal calf serum,glutamine and antibiotics. Fowlpox virus was purified by two successivesucrose gradients according to Joklik, W. K., Virology 18:9-18 (1962).Viral DNA was prepared by the proteinase K/SDS procedure according toGross-Bellard et al., Eur. J. Biochem. 36:32-38 (1973).

Construction of a fowlpox virus vector (f-TK2a) having a unique (NotI)cleavage site in an inserted DNA segment: The vaccinia virus tk-gene,together with the E. coli lacZ gene was inserted into the intergenicregion between the tk-gene and the 3′-orf of fowlpox virus. The plasmidspTKm-VVtka and pTKm-VVtkb were constructed by cloning the functionalvaccinia virus tk-gene into the intermediate plasmid pTKm-sP11. Uponintracellular recombination of pTKm-VVtka and pTKm-VVtkb with wildtypefowlpox virus DNA two novel FPV vectors, termed f-TK2a and f-TK2b,respectively, were created. Each vector contains two functionaltk-genes, the endogenous FPV gene and the inserted vaccinia virustk-gene, in addition to the inserted lacZ gene, any of which can be usedas a non-essential site for insertion of foreign DNA. In particular, theNotI site in the lacZ gene is a unique cleavage site in the f-TK2a and bvectors and, therefore, is advantageous for direct molecular cloning offoreign DNA into these vectors. Complete details of the construction ofthe fowlpox virus vectors f-TK2a and f-TK2b are disclosed in U.S. Ser.No. 07/935,313 entitled “Recombinant Fowlpoxvirus” by Dorner et al.,which claims priority of an equivalent European application filedconcurrently with this application, the entire disclosure of which ishereby incorporated herein by reference.

In vivo packaging in avian cells: 8×10⁶ CEF cells are infected with 0.2pfu/cell of helper virus (HP2) for 2 h. For packaging engineered FPVgenomes, 1 μg of purified ligation reaction product is used. Cells aretransfected with DNA's by the calcium phosphate precipitation techniqueand incubated for 15 min at room temperature. Graham and van der Eb(1973). Nine ml medium (TCM 199, 10% fetal calf serum, glutamine andantibiotics) per one ml precipitate are added to the cells. After fourhours the medium is changed and further incubated for two days. Crudevirus stocks are prepared according to standard procedures. Mackett etal. (1985). Plaque assays and gpt-selection are conducted as describedby Scheiflinger et al. (1991).

Direct molecular cloning into a unique NotI cleavage site of a fowlpoxvirus genome: The recombinant FPV strain f-TK2a is suitable as a vectorfor directly cloning a gene cassette, for instance a model gpt genecassette as described herein, into a unique NotI cleavage site.Scheiflinger et al. (1991). This NotI site of the vector is in thecoding region of a lacZ gene, which serves as a color screening markerthat is inactivated upon gene insertion. Thus, lacZ-positive virusesform blue plaques in the presence of the chromogenic substrate X-Gal,while viruses with inserts in this NotI site show a white plaquephenotype. The genome of the f-TK2a vector also has incorporated thevaccinia virus tk gene that also serves as an alternate gene insertionregion. Both the lacZ and tk genes were inserted into the fowlpox virusgenome in the intergenic region between the fowlpox tk gene and the3′-open reading frame, by conventional methods. Scheiflinger et al.(1991).

Patterns of DNA cleavage by NotI were established for the genomic DNA'sof FPV viruses HP1.441 and the vector strain f-TK2a (FIG. 2.1). HP1.441was derived from a virulent FPV strain through attenuation by serialpassage in chicken embryo fibroblasts. HP1.441 is the 441st passage ofHP1 and is used as a vaccine strain against fowlpox and is well adaptedfor rapid replication in cell culture. Mayr and Malicki (1966).

DNA from HP1.441 was analyzed as a reference for the FPV vector strainf-TK2a which is a derivative of HP1.441. The restriction analysis of theHP1.441 DNA (FIG. 2.1, lanes 1 and 2) showed that this strain has noNotI sites. Cleavage of vector f-TK2a DNA with NotI resulted in twolarge fragments of about 100 and 200 kb (FIG. 2.1, lane 4).

Direct molecular construction of a fowlpox virus expressing the gptgene: A model gene expression cassette comprising the E. coli gpt genewas constructed in the plasmid pN2-gpta which contains the gpt genedriven by an early/late poxvirus promoter flanked by NotI sites (FIG.1.3).

For cloning into the vector f-TK2a, the gpt gene cassette is excisedfrom its plasmid and ligated with NotI cleaved genomic DNA of f-TK2a asoutlined in FIG. 2.2. Ligated DNA is transfected into fowlpox helpervirus-infected CEF cells. gpt-positive plaques that remain white underan overlay containing X-Gal are further analyzed by Southern blotanalysis after infection of chicken embryo fibroblasts. Total cell DNAis isolated and the separated NotI fragments are subjected to Southernblot analysis with ³²P-labelled DNA's of the helper fowlpox (HP2) andgpt gene sequences, as described in Example 1. gpt-positive virusescontaining the gpt gene on the 1.1 kb NotI fragment indicating thatcorrect ligation has occurred in the cloning step.

Production of modified viruses with both insert orientations in oneconstruction step: The present example also illustrates how viruseshaving a single copy of the inserted gene cassette in eitherorientation, as well as viruses containing multiple copies of theinserted gene, can be recovered from a single direct molecular cloningstep. The orientation of the DNA insert in selected engineered fowlpoxgenomes is determined by Southern blot analysis of DNA's cleaved withappropriate restriction enzymes. As shown in FIG. 2.2, the DNA insertedinto a viral DNA may be in either the “a” or “b” orientations. Forpreliminary analyses of insert number and orientation with the presentmodel gene cassette, for instance, total DNA of cells infected withselected plaques is digested with the restriction endonuclease ClaI andNotI and separated on a 0.8% agarose gel. The blot is hybridized with agpt gene probe and a fowlpox virus probe.

In the NotI-digested DNA samples of recombinant viruses, the gptcassette is excised as a 1.1 kb fragment. Cleavage with ClaI of DNA'shaving an insert in the a or b orientation also results in differentcharacteristic fragments hybridizing with a gpt gene probe, asdetermined from the structures presented in FIG. 2.2.

EXAMPLE 3 Heterologous packaging of engineered orthopox (vaccinia) virusgenomic DNA by an avipox (fowlpox) helper virus and subsequent selectionfor recombinants in host cells of a species in which the helper viruscannot replicate

Heterologous packaging of poxvirus DNA, for instance, packaging of anorthopoxvirus DNA by an avipox virus, has not been reported. However,the present example demonstrates that in vivo packaging ofextracellularly engineered vaccinia virus DNA can be achieved by fowlpoxvirus in chicken embryo fibroblasts. The use of a vector virus having adifferent host range from that of the helper virus provides a simple andefficient procedure for purifying an engineered virus in one plaqueassay step. Thus, in the present example, the recombinant orthopoxviruswas recovered by plaque assay on mammalian (CV-1) cells which do notsupport full replication of the avipox helper virus. Inclusion of adominant selective marker in the DNA inserted into the vectoradvantageously facilitates the use of selective plaque assay conditionsfor elimination of viruses comprising vector DNA lacking the desiredinsert.

Another advantage of the heterologous packaging approach is the reducedpotential for recombination between vector and helper viruses. Forexample, orthopox and avipox viruses belong to different genera, havedifferent morphologies and replication facilities, and share onlyminimal sequence homology as demonstrated by a lack ofcross-hybridization under standard hybridization conditions. Therefore,homologous recombination of the genomes of avipox and orthopox virusesis exceedingly unlikely and use of these two viruses can practicallyeliminate undesirable recombination events that frequently occur betweenhomologous sequences of closely related viruses. Fenner and Comben,Virology 5:530-548 (1958); Fenner, Virology 8:99-507 (1959). Analternative approach for preventing vector-helper recombination duringpackaging is to use recombination deficient virus strains or host cells.

In this example, a model expression cassette comprising a marker gene(the E. coli gpt gene driven by a poxvirus promoter) was insertedextracellularly into a unique SmaI site of vaccinia virus DNA. The useof this restriction enzyme to cleave the viral DNA produces blunt endswhich advantageously may be ligated to blunt-ended DNA inserts preparedby any other nuclease that produces blunt ends, or, for example by usinga polymerase or exonuclease to create blunt ends from an insert havingsingle-stranded ends.

For packaging, the engineered genomic DNA was transfected into fowlpoxvirus-infected host cells in which both vaccinia and fowlpox viruses canreplicate (chicken embryo fibroblasts). Since the host range of thefowlpox helper virus is restricted to avian cells, vaccinia virus cloneswere selected by plaque-purification of progeny from the transfectedcells on mammalian host cells (African Green Monkey Kidney CV-1 cells).Simultaneous selection for gpt gene expression was used to isolation ofonly modified vaccinia viruses. In contrast to the conventional methodof producing poxvirus recombinants where in one intracellular geneticcross usually only one copy of a foreign gene can be inserted in asingle orientation, in the present example, both possible orientationsof a single insert, as well as double insertions of the model genecassette were identified as products of a single extracellular genomicmodification reaction.

The experimental results in the present example show that the packagingefficiency of ligated vaccinia virus DNA's by fowlpox helper virus waslow compared to packaging of intact vaccinia virus DNA with fowlpoxvirus, which produces yields in the range of 5×10³ to 1×10⁴ pfu per6×10⁶ chicken embryo fibroblasts after three days of replication. In onepackaging experiment (producing plaques designated the “F12” series,infra) the yield of packaged modified virus was 9×10² pfu, and in asecond experiment (producing the “F13” series), 5×10² pfu, per 6×10⁶chicken cells. One source of this relatively low packaging frequency inthese experiments is the lack of dephosphorylation treatment of thevector DNA arms which, therefore, were able to relegate efficientlywithout any insert. Such treatment was omitted because dephosphorylationof blunt-ended DNA fragments is usually inefficient. This problem can beovercome by construction of host virus strains having multiple cleavagesites with “sticky” ends that enable directional (“forced”) cloning,thereby making the insertion of foreign DNA fragments much moreefficient.

Another factor influencing the packaging efficiency is interference atthe cellular level between the helper and the packaged virus. Understandard packaging conditions, within three days of incubation thehelper virus (fowlpox) usually replicates to titers of about 1×10⁸ pfuper 6×10⁶ chicken embryo fibroblasts. The large excess of fowlpox viruscompared to packaged vaccinia virus creates conditions that producenegative interference phenomena and inhibits replication of the packagedvirus.

This interference is minimized by using mammalian cells for packaging incombination with fowlpox helper virus as described in Example 7. In thatcase, the host cells do not support full replication of the helperfowlpox virus. Although, no testing of ligated vaccinia virus DNA forpackaging efficiency by fowlpox virus has been made in a mammalian hostcell, a packaging yield of 2×10⁶ pfu per 8×10⁶ mammalian (CV-1) cellswas obtained with uncleaved vaccinia virus DNA.

In each viral recombinant generated by intracellular recombination witha given insertion plasmid an insert has one orientation depending on thepolarity of the homologous flanking regions in that plasmid. Due totranscriptional interference phenomena, for instance, expression levelsfor genes inserted into a poxvirus vector depend on the orientation ofthe foreign gene relative to the viral genome. Ink and Pickup (1989).Therefore, it is desirable to obtain in one reaction step modifiedviruses having either possible orientation. One of the advantages of theprocedure in this example is that both possible orientations of theinserted DNA are obtained in one ligation reaction, allowing immediatescreening for variants having the highest expression level. Thepreferred orientation of the cassette of this example in the selectedSmaI insertion site of vaccinia virus is the “b” orientation, asevidenced by the fact that the majority of modified viruses had thisgenomic structure. In this cassette the P7.5 promoter controlling theforeign gene is in the inverted repeat orientation relative to theendogenous 7.5 kDa polypeptide gene. As discussed in Example 1, theendogenous 7.5 kDa polypeptide genes are located in the invertedterminal repetitions of the vaccinia genome. The distance of the P7.5promoter of the gpt gene and the P7.5 promoter in the left terminalrepetition is about 20 kb. The “a” orientation should therefore be lessstable and less frequently obtained, in accordance with the observationthat this orientation was found only twice. However, the viral isolatesF13.4 (orientation a) and vF12.5 (orientation b) were propagated tolarge scale with gpt-selection and were found to have stable predictedstructures. The stability of the various structures comprising multipleinserts without selection remains to be determined.

The ligations contained several-fold excess of insert over the vector,thereby favoring insertion of multiple copies of the cassette asobserved. However, it is unclear why in this example double insertionswere more frequent than in Example 1. Due to internal recombinationevents only certain configurations of multiple inserts are expected tobe stable. Further studies to evaluate stability of viruses withmultiple inserts and the optimal ratio of vector to insert for stabilityand expression level which depends on copy number can all be conductedas necessary for each construct, according to the teachings of thisapplication.

Purification of virus and DNA: The viruses and methods of Examples 1 and2 were used.

Engineering of viral DNA: Viral DNA purified from virions was cleavedwith SmaI and purified by one phenol extraction and three chloroformextractions. In the first experiment below, 2 μg of cleaved virus DNAwere ligated with 400 ng (34 fold molar excess) of the insert fragment(the 1.1 kb HpaI-DraI fragment excised from plasmid pTKgpt-Fls) in avolume of 30 μl for 40 h with 15 units of T4 ligase (Boehringer, Inc.).The second ligation experiment was done under the same conditions exceptthat a seventeen-fold molar excess of the 1.1 kb SmaI insert and 5 unitsof ligase were used.

In vivo heterologous packaging in avian cells: Chicken embryofibroblasts (6×10⁶) infected with the helper virus (0.5 pfu/cell ofHP1.441) and incubated for 2 h. Two μg of ligated DNA was transfectedinto the infected cells and treated further as described for thehomologous packaging procedure in Example 1. The initial plaque assaywas done in CV-1 cells as described in Example 1.

Demonstration of packaging of modified vaccinia virus DNA by fowlpoxhelper virus: The design of this experiment is shown in FIG. 3.1.Vaccinia virus genomic DNA was prepared from sucrose gradient purifiedvirions, cut with the restriction endonuclease SmaI, and ligated withthe blunt-ended foreign gene cassette. Ligated DNA was transfected intofowlpox virus-infected chicken embryo fibroblasts for packaging. Progenyvirus was identified by plaque assay on mammalian (CV-1) cells which donot support complete replication of fowlpox virus to produce infectiousvirions.

In more detail, first, the HpaI-DraI fragment bearing the model genecassette (containing the gpt gene driven by the vaccinia virus P7.5promoter) was excised from the plasmid pTKgpt-F1s and ligated directlyinto the unique SmaI site of vaccinia wildtype virus (WR strain).Falkner and Moss (1988). The gpt gene was selected to permit positiveselection of modified viruses. Boyle and Coupar (1988); Falkner and Moss(1988). The single SmaI site in vaccinia virus DNA is located in theopen reading frame A51R in the HindIII A fragment of the genome. TheA51R gene is non-essential for viral replication in cell culture. Goebelet al. (1990).

Ligated material was transfected into chicken embryo fibroblastsinfected with fowlpox helper virus. After three days the cells wereharvested and a crude virus stock was prepared. Packaged vaccinia viruswas identified by plaque assay on an African Green monkey kidney cellline (CV-1) in medium that selects for cells infected with a viruscarrying the gpt gene. This selection scheme prevents viruses containingself-ligated wildtype vaccinia virus DNA from forming plaques whileallowing modified viruses containing an inserted model gpt gene cassetteto do so.

The packaging frequency was low in initial experiments. The titer ofgpt-positive vaccinia virus in the crude stock prepared from 6×10⁶chicken embryo fibroblasts was in the range of 1×10² to 1×10³ pfu.

Thirteen gpt-positive plaques were amplified under gpt-selection in CV-1cells. Total DNA of infected cells was isolated, digested with HindIII,separated on a 0.7% agarose gel and further processed for analysis bySouthern blot analysis with a gpt gene probe. As shown in FIG. 3.2,several viruses having blot patterns predicted for different modifiedgenomic structures were obtained.

In lanes 2, 4, 11 and 13 (corresponding to plaques #F12.3, F12.5, F13.3and F13.5) a single hybridizing fragment of about 45 kb is visible, thatis expected when one copy of the gene cassette is inserted into theviral genome in the “b” orientation into the viral genome (see FIG.3.3). An expected novel fragment of 5.2 kb is also present in all cases,and also appears when the same DNA's are tested as in FIG. 3.2 using avaccinia virus probe.

Two viruses having patterns consistent with the “a” orientation wereobtained in lanes 7 and 12 (corresponding to plaques #F12.8 and F13.4),where a single gpt-hybridizing fragment of about 5.7 kb is expected. The5.7 kb fragment in lane 7 is more visible in longer exposures of theautoradiograph. The pattern seen in lane 5 (plaque F12.6) may representa single insert in the “a” orientation, but the expected 5.7 kb band issomewhat larger for unknown reasons.

The pattern of three viral isolates is consistent with a tandeminsertion in the “a” orientation (lanes 1, 6 and 10, corresponding toplaques #F12.2, F12.7 and F13.2). In these cases two gpt-positivehybridizing fragments, of 5.7 and 1.1 kb, are expected (see also FIG.3.3). Fragments of 5.7 and 1.1 kb were also observed in equimolaramounts with the viral DNA in a blot hybridized with a vaccinia virusprobe.

The genome of the isolate in lane 3 (plaque F12.4) probably contains atandem duplicate insert in the “b” orientation. In this case twofragments, of 45 kb and 1.1 kb, are expected to hybridize with thegpt-gene.

The viral DNA in lane 9 (plaque F13.1) may comprise a head-to-headdouble insertion. In this case a 45 kb and a 5.7 kb fragment hybridizingwith a gpt-gene probe are expected. However, in addition such a DNAshould contain a novel 0.6 kb fragment that hybridizes with a vacciniaDNA probe, and, in fact, this fragment was detected on a blot hybridizedwith a vaccinia probe. Nevertheless, the expected 5.7 kb fragment wassomewhat smaller than predicted and produced a hybridization signal thatwas weaker than expected. Therefore, confirmation of the structure ofthis recombinant requires more detailed analysis.

Further analysis revealed that the viruses F12.7 and F12.3, interpretedabove as having double insertions with tandem ‘a’ structures, and thevirus F12.4, interpreted above as having double insertions with tandem‘b’ structures, actually have multiple tandem inserts in the ‘a’ or ‘b’orientations, respectively. The Southern blot analysis of FIG. 3.2 doesnot distinguish between double tandem and multiple tandem inserts.

EXAMPLE 4 Construction of an orthopoxvirus (vaccinia) vector (vdTK) witha directional master cloning site and plasmids with compatibleexpression cassettes

This example demonstrates application of the methods of the presentinvention to create novel poxvirus cloning vectors by direct molecularmodification and cloning of existing poxvirus genomes. In particular,this example describes a vaccinia virus vector (vdTK) which allowsdirectional insertion (i.e., “forced cloning”) of foreign genes into ashort “multiple cloning site” segment comprised of several differentendonuclease cleavage sites each of which is unique in the vectorgenome. Forced cloning eliminates the need for selection or screeningprocedures to distinguish the desired recombinants from vector viruslacking an insert because incompatibility of DNA ends cleaved bydifferent nucleases prevents religation of the vector arms without aforeign insert. Consequently, the forced cloning approach is the mostefficient way to insert a foreign gene into a viral vector.

The directional vector vdTK is created by inserting a multiple cloningsite (comprised of unique NotI, SmaI, ApaI and RsrII sites) in place ofthe tk gene of vaccinia virus (see FIG. 4.1A). This nonessential locusis the site most frequently used for insertion of foreign genes intovaccinia virus, mainly because positive selection for tk-negativeviruses is available. Thus, when ligated vdTK vector DNA is packaged bya tk-positive helper virus, the vector virus may be positively selectedfrom the excess of helper virus. Further, insertion of foreign DNA intothe vaccinia virus tk-locus by conventional methods generally results instable recombinants.

The multiple cloning site of the new vdTK vector is comprised of NotIand SmaI cleavage sites which are unique in the vector. Prior toinsertion of the multiple cloning site, NotI and SmaI cleavage sitespreexisting in the wildtype vaccinia virus (WR strain) are deleted bydirect molecular modifications according to the present invention.Viruses having the desired modifications are detected by screeningtechniques based on the polymerase chain reaction (PCR) method foramplification of specific nucleic acid sequences. This example alsodescribes a set of plasmids which facilitate expression of DNA'sencoding complete or partial open reading frames in the vdTK vacciniavector. The present invention comprehends insertion of open readingframes directly into a poxvirus expression vector having all appropriateregulatory elements suitably placed for expression of the inserted openreading frame. However, the instant vdTK vector is not equipped withsuch regulatory sequences for expression of an inserted open readingframe that lacks its own transcription and translation signals.Accordingly, the plasmids of this example provide convenient geneexpression cassettes for routine linkage of open reading frames topoxvirus promoters and, optionally, to a translation start codon. Anopen reading frame and associated regulatory sequences are thenefficiently transferred into the vdTK vector master cloning site byforced cloning. Modified viruses having the insert in either orientationcan be obtained by using one of two plasmids having the expressioncassette in the desired orientation within its master cloning site. Thegene expression cassettes of the plasmids exemplified here have twonested sets of restriction enzyme cleavage sites to facilitate cloningof open reading frames into the vdTK vector. The cassettes have a mastercloning site comprised of the same unique sites as the master cloningsite of the vdTK vector. In addition, in the middle of this mastercloning site the cassettes contain a variety of sites for frequentlycutting enzymes that are useful for insertion of open reading framesinto the cassettes. Thus, DNA's inserted into a cassette by means of thefrequent cutter sites are flanked on either side by several differentunique sites which are suitable for forced cloning of the cassette intothe master cloning site of the vdTK vector.

This example also describes gene expression cassettes suitable forinsertion into a single unique site in the vaccinia virus vector vdTK.To overcome the reduced cloning efficiency of using a single enzyme forcleaving the vector DNA, the expression cassettes of these plasmidsinclude the E. coli gpt gene as a selective marker.

The vdTK vaccinia vector system is preferentially used in conjunctionwith the heterologous packaging procedure described in Examples 3 and 7.The plasmids containing the gpt marker can also be used with homologoushelper virus lacking the gpt marker. Examples of constructs forexpression of polypeptides using the vdTK vector and related plasmidsystem are presented hereinbelow in Example 5.

In addition to the above advantages, the expression cassette plasmids ofthis invention also provide a means of overcoming a general problem ofincompatibility between the ends of cleaved poxvirus vector DNA's andmany insert DNA's, as a convenient alternative to the common use ofsynthetic adaptor DNA segments. Thus, isolation of DNA fragmentsencoding open reading frames usually is facilitated by use ofrestriction endonucleases having recognition sequences which are shortand, consequently, randomly occur at high frequencies in all natural DNAsequences. On the other hand, such frequently cutting enzymes generallyare not suitable for efficient direct cloning into genomes as large asthose of poxviruses, for instance, because such enzymes cleave largeDNA's into many fragments. Religation of these fragments would occur inrandom order, producing few intact viral genomes. Therefore, insertionsites in a vaccinia vector preferably are cleavage sites of infrequentlycutting restriction endonucleases which are unlikely to be used forisolation of open reading frame fragments or insert DNA's in general.The present plasmids overcome this general incompatibility by allowingefficient insertion of fragments from frequent cutters into the plasmidfollowed by efficient transfer into the vaccinia vector usinginfrequently cutting enzymes.

Deletion of the unique NotI cleavage site from wildtype vaccinia (WR)virus: The unique NotI site of vaccinia virus may be eliminated byinsertion into this site of a “NotI deletion adaptor” segment havingcohesive ends compatible for ligation with NotI-cleaved DNA but lackingsequences required for recognition by the NotI endonuclease. Thus, thesequences formed by the ligated cohesive ends of the NotI-cleaved viralDNA and viral DNA and adaptor are not cleavable by NotI. This adaptoralso contains several selected restriction endonuclease cleavage sitesfor directed insertion of DNA fragments.

More particularly, one μg of vaccinia virus WR wild type DNA is cut withNotI and ligated with one μg of the double-stranded NotI-deletionadaptor. The adaptor consists of two partially complementary strands:odN1 (SEQ ID NO:16) and odN2 (SEQ ID NO:23). The central part of theadaptor contains the restriction endonuclease cleavage sites StuI, DraI,SspI and EcoRV. Annealed adaptor oligonucleotides are used for theligation reaction. The ligated material is transfected into fowlpoxvirus-infected chicken embryo fibroblasts and packaged as described inExample 3.

An alternative procedure for deleting the single NotI site of vacciniavirus (WR strain) is outlined in FIG. 4.1B. In the first step, vacciniavirus DNA is cut with SacI, the SacI “I” fragment is isolated from lowmelting point agarose and cloned into the SacI site of a suitableplasmid, such as pTZ19R (obtainable from Pharmacia, Inc.). The resultingplasmid, pTZ-SacI, is cut with NotI, treated with Klenow polymerase tofill in the sticky ends and religated. The ligated material istransfected into E. coli cells (HB101). The colonies are isolatedaccording to standard cloning procedures. The resulting plasmid,pTZ-SacIdN has the NotI site deleted and is used in a reversegpt-selection experiment as described by Isaacs et al., Virology178:626-630 (1990), modified as follows.

CV-1 cells (8×10⁶) are infected with 0.2 pfu of the viral isolate vp7, avaccinia virus that has integrated into the single NotI site a gpt genecassette (see Example 1). Subsequently, a calcium-phosphate precipitatecontaining 20 μg of DNA from the modified SacI fragment prepared fromthe plasmid pTZ-SacIdN is transfected into the cells. The cells arefurther treated as described in the packaging procedure in Example 1.Crude virus stocks are used to infect mouse STO cells (obtained from theAmerican Type Culture Collection, Rockville, MD; ATCC# CRL 1503) in thepresence of 6-thioguanine (6-TG). This is a negative selection procedurethat requires the loss of the gpt gene for a virus to replicate and,therefore, leads in the present case to integration of the modified SacI“I” fragment and, thereby, deletion of the gpt gene. See Isaacs et al.(1990). All plaques growing in the presence of 6-TG should lack the gptgene and contain a modified SacI I fragment. The estimated yield is inthe range of 0.1-0.2% of the total plaques (i.e., the normal frequencyof recombinants in this type of marker rescue experiment). Since theselection procedure is extremely efficient identification of the correctstructures is not expected to require examination of large numbers ofclones. See Isaacs et al. (1990) However, whether the first procedureabove or this alternative procedure is used to delete the single NotI ofvaccinia virus, the following screening procedure may be used toidentify the desired construct.

Identification by PCR-screening of virus (vdN) having the NotI sitedeleted: Vaccinia virus clones having the NotI site deleted may beidentified by analysis of plaques growing in a cell line (CV-1) thatdoes not support the growth of the fowlpox helper virus. The DNA's ofviruses in individual plaques are analyzed by a PCR-based screeningmethod, as follows.

The first primer for the PCR reaction is the oligonucleotide odn1, (SEQID NO:16), and the second primer was odN3 (SEQ ID NO:24). The sequenceof second primer is located in the vaccinia virus genome about 770 bpdownstream of the first primer sequence. The template is total DNA from1×10⁶ CV-1 cells infected with half the virus of a single plaque. DNA isprepared by standard techniques and about 50 ng is used for the PCRreaction. The PCR reactions are carried out according to standardtechniques using commercially available PCR kits. Positive PCR reactionsproduce a DNA fragment of about 770 bp. Such a virus having the NotIsite deleted is designated “vd^(N”.)

Deletion of the unique SmaI restriction site from vaccinia virus vdN:The WR strain of vaccinia virus contains a single SmaI site in an openreading frame (A51R) which is not essential for virus replication incell cultures. Goebel et al. (1990). Although this site may be used forforeign gene insertion, in the present example, however, this site isdeleted in favor of creating a more versatile vaccinia virus vector byintroducing a new unique SmaI site as part of a multiple cloning sitecassette.

Accordingly, vdN virus DNA (1 μg) is cut with SmaI and ligated with anexcess of a hexamer linker having the recognition sequence for therestriction nuclease HindIII (odS1, 5′-AAGCTT-3′). Insertion of thislinker into the vaccinia virus SmaI cleavage site results in destructionof the SmaI recognition sequence and the introduction of a new HindIIIrecognition sequence. The ligated material is packaged by transfectioninto cva cells that have been infected with fowlpox virus, as describedin Example 7.

Alternatively, the single SmaI site of vaccinia virus (WR strain) isdeleted according to the procedure outlined in FIG. 4.1C, by modifying acloned fragment of vaccinia virus DNA instead of directly modifying thecomplete vaccinia virus DNA. In a first step, vaccinia virus DNA is cutwith SalI, the SalI F-fragment is isolated from low melting pointagarose and cloned into the SalI site of a suitable plasmid, such aspTZ9R (obtainable from Pharmacia, Inc.). The resulting plasmid,pTZ-SalF, has two SmaI sites, one in a multiple cloning site and theother in the vaccinia sequences (FIG. 4.1C). pTZ-SalF is partiallydigested with SmaI and I-SceI linkers are added, as follows: firststrand, I-SceI linker 1 (SEQ ID NO:25) and its complementary strand,I-SceI linker 2 (SEQ ID NO:26). The correct plasmid having the SmaI sitedeleted from the vaccinia sequences is identified by cleavage with SmaIand I-SceI. The final plasmid, pTZ-SalFdS, is used to introduce the SmaIdeletion into a vaccinia virus genome using the reverse gpt geneselection experiment as described for deletion of the NotI site, exceptthat preferred virus to be modified is the isolate F12.5, a virus thathas integrated into the single SmaI site a gpt gene cassette (seeExample 3).

The resulting insertion of a site for endonuclease I-SceI advantageousfor direct molecular cloning because this enzyme, isolated from yeast,recognizes an 18mer site and, therefore, cuts random DNA sequencesextremely infrequently. For instance, I-SceI cuts the yeast genome onlyonce. Thierry et al., Nucleic Acids Res. 19:189-190 (1991). I-SceI iscommercially available from Boehringer, Inc. Advantageously, an I-SceIsite is introduced into a vector having no preexisting sites for thatenzyme, thereby creating a new vector with a single site that can beused for gene insertions. Whether a vaccinia virus DNA or other vectorDNA contains a site for I-SceI cleavage can be determined by routinerestriction analyses of the vector DNA.

Where this alternative procedure for deletion of the SmaI site fromvaccinia virus DNA is used, the order of steps for constructing thevector vdTK is as follows: deletion of the SmaI site resulting in virusvdS (see above); deletion of the NotI site by insertion of the NotI gptgene cassette (see Example 1) into the single NotI site of vdS bycloning and packaging, resulting in the virus vdSNgpt and reversegpt-selection as described above, using vdSNgpt and pTZ-SacIdN assubstrates for the marker rescue experiment; and deletion of the tk geneas outlined in below in the present example.

An alternative procedure by which the vector vdTK actually wasconstructed is as follows. The SmaI site of vaccinia wild-type virus wasdeleted, creating the intermediate virus vdS. In a second experiment theNotI site was deleted from vaccinia wild-type virus creating theintermediate virus vdN. The virus vdSN was obtained by co-infectionusing both viruses of CV-1 cells and PCR screening of the recombinantvirus (that was created by a simple genetic cross-over event). Theviability of the different intermediates was determined by titrations.

Table 2 at section A shows the results after individual isolates fromthe vdN cloning experiment were plaque purified five times (to insurethat wildtype virus-free clones were obtained) and then amplified. Aftertitration, crude virus stocks of the first amplification, together withwild-type control (WR-WT), were used to infect CV-1 cells at 0.1pfu/cell. These cells were harvested after 48 h and used to preparecrude stocks which were re-titered. These results are shown in Table 2at section B. Isolates vdN/A1 #6.1111 and vdN/A1 #10.1111 weredesignated as clones vdN#6 and vdN#10, respectively, and used for largescale virus preparations.

Table 3 at section A shows the results after single isolates of the vdScloning experiment were plaque purified five times and then amplifiedand titered. Crude stocks of the first amplification, together withwild-type control (WR-WT), were used to infect CV-1 cells at 0.1pfu/cell. The cells were harvested after 48 hours and the resultingcrude stocks were re-titered. These results are shown in FIG. 4.2 atsection B. The isolates vdS# 7.11 were designated as clones vdS#2 andvdS#7, respectively, and used for large scale virus preparations. Ineach case, the virus isolate showing the best growth characteristics wasselected to be amplified and grown to large scale.

TABLE 2 Viability Studies of the Viral Intermediate vdN A) Titer afterfirst amplification of six viral vdN-isolates (pfu/ml crude stock):vdN/A1# 2.1111 1.0 × 10⁷ pfu/ml vdN/A1# 4.1111 1.3 × 10⁸ pfu/ml vdN/A1#6.1111 9.0 × 10⁷ pfu/ml vdN/A1# 8.1111 8.0 × 10⁷ pfu/ml vdN/A1# 10.11114.0 × 10⁷ pfu/ml vdN/A1# 12.1111 1.1 × 10⁸ pfu/ml B) Titer after secondamplification: vdN/A1# 2.1111 3.6 × 10⁸ pfu/ml vdN/A1# 4.1111 2.5 × 10⁸pfu/ml vdN/A1# 6.1111 5.9 × 10⁸ pfu/ml vdN/A1# 8.1111 4.2 × 10⁸ pfu/mlvdN/A1# 10.1111 4.3 × 10⁸ pfu/ml vdN/A1# 12.1111 2.2 × 10⁸ pfu/ml WR-WT5.4 × 10⁸ pfu/ml

TABLE 3 Viability Studies of the Viral Intermediates vdS A) Titer afterfirst amplification of five viral vdS-isolates (pfu/ml crude stock) vdS#2.11 4.1 × 10⁷ pfu/ml vdS# 3.11 6.5 × 10⁷ pfu/ml vdS# 4.11 8.0 × 10⁷pfu/ml vdS# 5.11 2.7 × 10⁷ pfu/ml vdS# 7.11 4.7 × 10⁷ pfu/ml B) Titerafter second amplification vdS# 2.11 1.6 × 10⁸ pfu/ml vdS# 3.11 1.4 ×10⁸ pfu/ml vdS# 4.11 8.0 × 10⁷ pfu/ml vdS# 5.11 1.3 × 10⁸ pfu/ml vds#7.11 1.7 × 10⁸ pfu/ml WR-WT 2.8 × 10⁸ pfu/ml

Identification by PCR-screening of virus (vdSN) having the SmaI sitedeleted: Clones of the vdSN vaccinia virus having the SmaI site deletedare identified by PCR screening as follows. The first primer for the PCRreaction is the oligonucleotide odS2 (SEQ ID NO:27) and the secondprimer is the oligonucleotide odS3 (SEQ ID NO:28). The sequence ofoligonucleotide odS2 is located in the vaccinia genome about 340 bpupstream of the SmaI site, while that of oligonucleotide odS3 is locatedabout 340 bp downstream of this site. The template is total DNA of CV-1cells infected with a virus plaque as described above for vdNidentification. The PCR-amplified band of about 680 bp is tested forsusceptibility to SmaI, with resistance to SmaI cleavage indicatinginsertion of the HindIII or I-SceI linker, while wildtype control DNA iscut into two pieces of about 340 bp. A vaccinia virus having the desiredinsertion of a linker in the SmaI site is designated vdSN.

Deletion of the coding region of the thymidine kinase gene from vacciniavirus vdSN: From vaccinia virus vdSN, a novel vector strain (designatedvdTK) is developed by replacing the tk gene, which is located in agenetically stable region of the vaccinia genome, with a segmentcomprised of several unique restriction endonuclease cleavage sites(FIG. 4.lA).

The tk coding sequence is first deleted from a plasmid (pHindJ-1)comprising a segment of the vaccinia genome (the HindIII J segment) inwhich the tk gene is located (see FIG. 4.2). In place of the tk-gene, amultiple cloning site with the unique sites NotI, SmaI, ApaI and RsrII,flanked by SfiI sites is then inserted. Finally, the modified virussegment is transferred into the vaccinia virus genome vdSN which wasthen designated vdTK (FIG. 4.1A). To further facilitate forced cloning,each of the two SfiI sites also may be made unique in the vector byexploiting the variable nature of the SfiI recognition sequence(GGCCNNNNNGGCC, SEQ ID NO:85). The sequences of two SfiI sites are asfollows: SfiI(1), GGCCGGCTAGGCC (SEQ ID NO:29) and SfiI(2),GGCCATATAGGCC (SEQ ID NO:30). This plasmid containing the finalmodification of the tk gene (pHindJ-3) is constructed from precursorplasmid pHindJ-1 by loop-out mutagenesis, and deletion of the tk gene isconfirmed by sequence analysis.

Construction of precursor plasmid pHindJ-1: Vaccinia wildtype virus DNAwas cut with HindIII and the resulting fragments were separated on a0.8% low melting point agarose gel. The HindIII J fragment was excisedunder UV-light and prepared according to standard techniques. Thefragment was inserted into the single HindIII site of the plasmid pTZ19R(Pharmacia, Inc.) resulting in pHindJ-1.

Construction of plasmid pHindJ-2: Plasmid pHindJ-1 is transfected intoE. coli strain NM522 and single-stranded DNA is prepared bysuperinfection with the helper phage M13K07 according to the protocolsupplied by Pharmacia. The single-stranded DNA serves as the templatefor site directed mutagenesis with the primer odTK1 (SEQ ID NO:31). Thisprimer is complementary to the promoter region and the region around thetranslational stop codon of the tk-gene. In its central part it containsthe unique restriction sites BamHI, HpaI, NruI and EcoRI. Themutagenesis procedure is carried out with a mutagenesis kit provided byAmersham, Inc., according to the manual provided by the supplier.

For construction of pHindJ-2, the tk-gene sequence has been described inWeir and Moss, J. Virol. 46:530-537 (1983). The tk-gene sequence isaccessible in the EMBL Data Library under the identifier (ID) PVHINLJ.The sequence of the vector part (pTZ19R) of the plasmid is availablefrom Pharmacia, Inc. The sequence around the deleted vaccinia virus tkgene in the plasmid pHindJ-2 is shown in SEQ ID NO:4. The 5′ region ofthe tk gene (bases #1-19 in the present listing; bases #4543-#4561 in IDPVHINLJ) is followed by the unique restriction sites BamHI, HpaI, NruIand EcoRI and the 3′ region of the tk gene (bases #44-#67 presentlisting; bases #5119-#5142 in ID PVHINLJ). Bases #4562 to 5118 in IDPVHINLJ, which contain part of the tk promoter and the tk gene codingregion, are deleted in pHindJ-2.

Construction of the plasmid pHindJ-3: Plasmid pHindJ-2 is digested withBamHI and EcoRI and a double-stranded linker containing the uniquerestriction sites NotI, SmaI, RsrII and ApaI, flanked by SfiI sites isinserted. The linker consists of oligonucleotides P-J(1) (SEQ ID NO:32)and P-J(2) (SEQ ID NO:33).

The modified sequence of pHindJ-3 is shown in SEQ ID NO:5. The insertedmultiple cloning site corresponds to oligonucleotide P-J(1). Theinserted sequence starts at position 21 and ends at position 99. Theflanking sequences are the same as described in pHindJ-2, supra.

To insert the tk-deletion into vaccinia virus, plasmid pHindJ-3 isdigested with HindIII and a shortened HindIII J fragment having atk-gene deletion is used for a marker rescue experiment. Sam and Dumbell(1981). Viruses having the tk gene deleted are isolated by tk negativeselection and identified by subsequent PCR screening.

More particularly, the modified HindIII fragment present in pHindJ-3 isexcised with HindIII and isolated with a low melting point agarose gel.The marker rescue is performed essentially as described by Sam andDumbell (1981) with the following modifications. 5×10⁶ CV-1 cells areinfected with 0.2 pfu per cell of vaccinia virus vdSN. After one hour ofincubation, one ml of a calcium-phosphate precipitate containing 1 μg ofthe modified HindIII J fragment is transfected into the infected cells.After two days growth a crude virus stock is prepared as described inExample 1 and titrated on human 143B tk-negative cells in the presenceof bromodeoxy-uridine (BrdU) as described by Mackett et al. (1982).tk-negative plaques may be further analyzed by PCR screening.

Identification of the thymidine kinase deletion virus (vdTK) byPCR-screening: The first primer for the PCR reaction is oligonucleotideodTK2 (SEQ ID NO:34), the sequence of which is located about 300 bpupstream of the tk gene. The second primer, odTK3 (SEQ ID NO:35), islocated about 220 bp downstream of the stop codon of the tk gene. Thetemplate is total DNA of CV-1 cells infected with a virus plaque, asdescribed for vdN screening. The amplification product resulting fromvirus having the tk gene deletion is about 520 bp, while the wildtypecontrol produces a fragment of about 1.1 kb.

Construction of plasmids comprising gene expression cassettes fortransfer to the vdTK vector: The plasmid pA0 is the basic plasmid thatcontains a master cloning site comprised of the unique sites of themaster cloning site of the vdTK vaccinia virus vector. Plasmid pA0 wasconstructed by replacing the multiple cloning site of a commerciallyavailable plasmid with a segment comprised of the unique sites of thevdTK vector and an XhoI site, as illustrated in FIG. 4.3.

More in particular, to delete the multiple cloning site of thepBluescript II SK- phagemid (Stratagene), the plasmid was digested withSacI and Asp718. The large vector fragment was ligated with an adaptorconsisting of the annealed oligonucleotides P-A(0.1) (SEQ ID NO:36) andP-A(0.2) (SEQ ID NO:37).

The multiple cloning site of pA0 (corresponding to the oligonucleotideP-A(0.1)) and twenty bases of the 5′- and 3′-flanking regions ofpEluescriptII SK- are shown in SEQ ID NO:6. The insert starts atposition 21 and ends at position 95. (The first “A” residue at the5′-end corresponds to position number 2187, the last “G” residue at the3′-end corresponds to position number 2301 of the plasmid pA0).

Construction of the plasmids pA1 and pA2: The plasmids pA1 and pA2(compriisng SEQ ID NO:8) were designed for insertion of DNA segments,e.g., synthetic or natural promoter fragments. They were constructed byinserting into the Xhol site of pA0 a linker comprising a secondmultiple cloning site of frequently cutting enzymes that do not cleavepA0. Both plasmids have the same structure except for the orientation ofthe second multiple cloning site (FIG. 4.3).

The pA0 plasmid was digested with XhoI and ligated with an adaptorconsisting of the annealed oligonucleotides P-A(1.1) and P-A(1.2).Plasmids of both possible orientations of the adaptor were isolated anddesignated pA1 and pA2.

The multiple cloning site of pA1 (corresponding to the oligonucleotideP-A(1.1)) and twenty bases of the 5′- and 3′-flanking regions of pA0 areshown in SEQ ID NO:7. The insert starts at position 21 and ends atposition 83. (The first “C” residue at the 5′-end corresponds toposition number 2222, the last “C” residue at the 3′-end corresponds toposition number 2324 of the plasmid pA1).

The multiple cloning site of pA2 (corresponding to the oligonucleotideP-A(1.2)) and twenty bases of the 5′ and 3′-ends of pA2 are shown in SEQID NO:10. The insert starts at position 21 and ends at position 195.(The first “C” residue at the 5′-end corresponds to position number2252, the last “G” residue at the 3′-end corresponds to position number2466 of the plasmid pA2-S1).

Construction of plasmids pA1-S1 and pA2-S1: Plasmids pA1-S1 and pA2-S1provide the strong synthetic poxvirus promoter S1 (bases 21-194 of SEQID NO:9), including a translational start codon, followed by a singleEcoRI site suitable for insertion of open reading frames that do nothave an associated start codon. Promoter S1 is a modified version of astrong poxvirus late promoter designated P2.

Plasmids pA1-S1 and pA2-S1 are obtained by inserting a firstdouble-stranded promoter fragment into the NdeI and BamHI site of pA1 orpA2, respectively, by forced cloning (FIG. 4.4A) In particular, vectorpA1 is digested with NdeI and BamHI and ligated with an adaptorconsisting of the annealed oligonucleotides P-P2m1.1 and P-P2m1.2. Theresulting plasmid is designated pA1-S1.

The synthetic promoter sequence of pA1-S1 (corresponding to theoligonucleotide P-P2m1.1) and twenty bases of the 5′- and 3′-flankingregions of pA1 are shown in SEQ ID NO:9. The insert starts at position21 and ends at position 193. (The first “C” residue at the 5′endcorresponds to position number 2228, the last “G” residue at the 3′endcorresponds to position number 2440 of the plasmid pA1-S1).

The vector pA2 was digested with NdeI and BamHI and ligated with anadaptor consisting of annealed oligonucleotides P-P2m1.1 and P-P2m1.2,as for pA1-S1, above. The resulting plasmid is designated pA2-S1.

The synthetic promoter sequence of pA2-S1 (corresponding to theoligonucleotide P-P2m1.2) and twenty bases of the 5′- and 3′-flankingregions of pA2 are shown in SEQ ID NO:10. The insert starts at position21 and ends at position 195. (The first “C” residue at the 5′endcorresponds to position number 2252, the last “G” residue at the 3′endcorresponds to position number 2466 of the plasmid pA2-S1).

Construction of plasmids pA1-S2 and pA2-S2: The plasmids pA1-S2 andpA2-S2 contain the strong synthetic promoter S2 (bases 21-73 of SEQ IDNO:11), a modified version of a strong late synthetic poxvirus promoterdescribed by Davison and Moss, J. Mol. Biol. 210:771-784 (1989). Theseplasmids do not provide a translational start codon with the promoterand, therefore, are suited for insertion of complete open reading framesthat include a start codon. The promoters have different orientationswith respect to the vdTK master cloning site in these two plasmids.

Plasmids pA1-S2 and pA2-S2 are obtained by forced cloning of a seconddouble-stranded promoter fragment into the HpaI and EcoRI sites of pA1and pA2, respectively (FIG. 4.5A). More particularly, plasmid pAl isdigested with the enzymes HpaI and EcoRI, and ligated with a syntheticlinker sequence consisting of annealed oligonucleotides P-artP(5) andP-artP(6). The resulting plasmid is designated pA1-S2.

The synthetic promoter sequence of pA1-S2 (corresponding to theoligonucleotide P-artP(5) and twenty bases of the 5′- and 3′-flankingregions of pA1 are shown in SEQ ID NO:11. The insert sequence starts atposition 21 and ends at position 68. (The first “T” residue at the5′-end corresponds to position number 2240, the last “A” residue at the3′-end corresponds to position number 2327 of the plasmid pA1-S2).

Similarly, the plasmid pA2 is digested with the enzymes HpaI and EcoRI,and ligated with the annealed oligonucleotides P-artP(5) and P-artP(6)as for pA1-S2. The resulting plasmid is designated pA2-S2. The syntheticpromoter sequence of pA2-S2 (corresponding to the oligonucleotideP-artP(6) and twenty bases of the 5′- and 3′-flanking regions of pA2 areshown in SEQ ID NO:12. The insert starts at position 21 and ends atposition 72. (The first “T” residue at the 5′-end corresponds toposition number 2263, the last “A” residue at the 3′-end corresponds toposition number 2354 of the plasmid pA2-S2).

After insertion of an open reading frame into any of the plasmidspA1-S1, pA2-S1, pA1-S2 or pA2-S2, the entire expression cassette can beexcised and inserted by forced cloning into corresponding sites in thevirus vector vdTK. The cassette can be inserted into the virus genome ineither orientation depending on the cloning plasmid used.

Construction of plasmids comprising expression cassettes with aselective marker (pN2gpt-S3A and pN2gpt-S4): Besides plasmids designedfor forced cloning, described hereinabove, two additional plasmids wereconstructed for transferring genes into one unique (NotI) site in apoxvirus vector with the help of the E. coli gpt selectable marker gene.They also provide two additional poxvirus promoters besides the S1 andS2 promoters described hereinabove.

The plasmid pN2gpt-S3A (FIG. 4.7) can be used to insert open readingframes lacking their own initiation codon. The genes to be transferredinto vaccinia virus (the gpt marker and the open reading frame) can beexcised either with NotI alone or with two enzymes, for example, NotIand SmaI (or RsrII or ApaI). The excised fragment is then inserted intothe corresponding site(s) of the virus vector vdTK.

The plasmid pN2gpt-S4 (FIG. 4.7) can be used to insert complete openreading frames including an AUG translation start codon. The cassettesconsisting of the gpt marker gene and the open reading frame can beexcised as described for pN2gpt-S3A. The promoters S3A (bases 21-107 ofSEQ ID NO:13) and S4 (bases 21-114 of SEQ ID NO:14) are modifiedversions of strong poxvirus late promoters.

These plasmids were constructed by first making plasmids pN2-gpta andpN2-gptb (FIG. 4.6) which contain an E. coli gpt gene driven by thevaccinia virus P7.5 promoter, flanked by several unique restrictionsites including NotI (FIG. 1.3). Insertion of the S3A or S4promoter-fragment into the unique PstI and ClaI sites in pN2-gptbresulted in the plasmids pN2gpt-S3A and pN2gpt-S4.

Construction of plasmids pN2-gpta and pN2-gptb: See Example 1 and FIG.4.6.

Construction of plasmid pN2gpt-S3A: The parental plasmid pN2-gptb wasdigested with PstI and ClaI and ligated with a synthetic linker sequenceconsisting of the oligonucleotides P-artP(7) and P-artP(8) (SEQ IDNO:40). The resulting plasmid was designated pN2gpt-S3A.

The synthetic promoter sequence of pN2gpt-S3A (corresponding to theoligonucleotide P-artP(7)) and twenty bases of the 5′- and 3′-flankingregions of pN2-gptb are shown for pN2gpt-S3A in SEQ ID NO:13. Theinserted DNA sequence starts at position 21 and ends at position 107.(The first T-residue at the 5′-end corresponds to position number 3328,the last A-residue at the 3′-end to position number 3454 of the plasmidpN2gpt-S3A).

Construction of plasmid pN2gpt-S4: The plasmid pN2-gptb was digestedwith PstI and ClaI and ligated with an adaptor sequence consisting ofthe oligonucleotides P-artP(9) and P-artP(10) (SEQ ID NO:41). Theresulting plasmid was designated pN2gpt-S4.

The synthetic promoter sequence of pN2gpt-S4 (corresponding to theoligonucleotide P-artP(9)) and twenty bases of the 5′- and 3′-flankingregions of pN2-gptb are shown for pN2gpt-S4 in SEQ ID NO:14. Theinserted DNA sequence starts at position 21 and ends at position 114.(The first “T” residue at the 5′-end corresponds to base #3328, the last“A” residue at the 3′-end to position base #3461 of the plasmidpN2gpt-S4).

EXAMPLE 5 Expression of polypeptides in a vaccinia virus vector (vdTK)by direct molecular insertion of gene expression cassettes

This example demonstrates the facility with which cloned genes can beinserted into a vaccinia virus vector (vdTK) of the present inventionfor rapid creation of poxvirus expression constructs using directmolecular insertion of gene expression cassettes described in Example 4.Here, use of the vdTK vector-cassette system to make constructs forexpressing several particular model polypeptides is described, includinghuman blood proteins (prothrombin and variants of plasminogen) and ahuman virus antigen (HIV gp160).

Construction of a modified vaccinia virus (vPTI) expressing humanprothrombin: Human prothrombin (PT) serves as a model for foreignprotein expression in a vaccinia virus vector of the present invention.A cDNA encoding prothrombin has been shown previously to be expressibleby a conventionally constructed recombinant vaccinia virus, as disclosedin PCT Application PCT/EP91/00139 by Falkner et al. (“the Falknerapplication”), the entire disclosure of which is hereby incorporatedherein by reference.

A modified prothrombin cDNA is excised as a 2.0 kb EcoRI fragment fromthe plasmid pTKgpt-PTHBb, and inserted into the single EcoRI site of theplasmid pA1-S1 (Example 4, FIG. 4.4A) resulting in the plasmid pA1S1-PT(FIG. 5.1). In the expression cassette of this plasmid, the prothrombincDNA is driven by the synthetic poxvirus promoter S1 which also providesa translation initiation codon.

The sequence of human prothrombin has been published by Degen et al.,Biochemistry 22:2087-2097 (1983). This sequence is accessible in theEMBO Data Library under the Identifier (ID) HSTHR1. The sequence in IDHSTHR1 is not complete; it lacks the first 19 bp of the prothrombincoding region. The present inventors have sequenced the missing part ofthe cDNA in ID HSTHR1 and present this hereinbelow.

Due to the many modifications and base changes, the full sequence of thepresent human prothrombin cDNA clone including the S1 promoter and 20bases of plasmid flanking sequences is shown in SEQ ID NO:15.

By the engineering steps outlined in the Falkner application, the cDNAwas modified as follows: two additional codons (bases #22-27) wereintroduced resulting in the incorporation of two new amino acids; the3′-untranslated sequence was removed by introduction of an EcoRI site:bases #1963-1965 (#1920-1922 ID HSTHR1) were changed from TGG to GAA bysite directed mutagenesis.

One base pair change was found in the present PT-cDNA, that results in anovel NcoI site: base #525 (#482 in ID HSTHR1) is changed from C to A.This is a silent mutation because the CCC codon (Pro) is changed to CCA(Pro) which results in a new NcoI site. (The first base of SEQ ID NO:15from pA1S1-PT corresponds to base #2394 and the last base to #4381 ofthe full sequence of plasmid pA1S1-PT).

For transfer into the vaccinia virus vector vdTK, the cassette isexcised from the plasmid pA1S1-PT with NotI and RsrII endonucleases andisolated after separation on a low melting point agarose gel. The virusvector vdTK DNA is cleaved with NotI and RsrII, extracted with phenoland precipitated with ethanol. The small NotI-RsrII connecting fragmentof the multiple cloning site of the vector DNA is lost during theethanol precipitation step. The vaccinia vector arms are ligated with atwenty-fold molar excess of cassette. Packaging of ligated vacciniavirus DNA with fowlpox helper virus in chicken cells is described inExample 3. Packaged viruses from plaques produced by infection of inCV-1 cells are plaque purified again and small crude stocks areprepared. The virus isolates may be further analyzed by Southern blotanalysis and expression analysis as described in the Falknerapplication. A viral isolate having the correct genomic structure forinsertion of the prothrombin cDNA is designated vPT1. A similarrecombinant vaccinia virus produced by marker rescue induced prothrombinexpression in Vero cells at levels of activity of about 50-60 mU/ml ofcell culture supernatant. See the Falkner application.

Construction of a vaccinia virus (vGPg1) expressing humanglu-plasminogen: The native form of plasminogen (Pg) has an aminoterminus starting with the amino acid glutamic acid (glu) and istherefore called glu-plasminogen (glu-Pg). A partially processed form ofplasminogen that lacks the first 77 amino terminal amino acids (theactivation peptide) is called lys-plasminogen (lys-Pg). The affinity oflys-Pg for its substrate fibrin is much higher than that of glu-Pg. Inaddition, recombinant lys-Pg is considerably more stable than glu-Pg insupernatants of cell cultures infected with a (conventional) vacciniarecombinant carrying the glu-Pg gene.

The complete human plasminogen cDNA (including its translational startand stop codons) was excised from a plasmid (phPlas-6) as a BalI-SmaIfragment. The sequence of human plasminogen has been published byForsgren et al., FEBS Letters 213:254-260 (1987), and is accessible inthe EMBO Data Library (GenBank) under the Identifier (ID) HSPMGR.Therefore sequences of this plasmid have not been included in theinstant Sequence Listing because this plasmid is not a unique source ofthe plasminogen DNA sequence. However, the coding region of the presentplasminogen sequence differs from the published sequence in at least onenucleotide: the “A” residue at position #112 (ID HSPMGR) is a “G”residue in the instant DNA, resulting in an amino acid substitution(Lys→Glu).

The plasminogen cDNA was inserted into the HpaI site of the plasmidpN2gpt-S4 (Example 4, FIG. 4.6), which was selected for constructing agene expression cassette with a selectable marker because theplasminogen cDNA contains two ApaI sites and one RsrII site andtherefore does not allow the use of the expression cassettes designedfor forced cloning. The resulting plasmid was designated pN2gpt-GPg(FIG. 5.2).

The joining region of the S4 promoter including the initiation codon ofplasminogen (base #32 this listing; base #55 in ID HSPMGR) is shown forpN2gpt-GPg in SEQ ID NO:17. The coding region of glu-plasminogen wasomitted in the sequence listing. The sequence continues with the stopcodon (base #35 this listing; base #2485 in ID HSPMGR) and 25 bases ofthe 3′-untranslated plasminogen sequence. This sequence is followed by29 bases of the multiple cloning site of phPlas6 and by 20 bases of themultiple cloning site of plasmid pN2gpt-S4.

To transfer the glu-plasminogen gene cassette into a vaccinia virusgenome, the NotI fragment of pN2gpt-GPg containing the two genes andtheir promoters (the P7.5 promoter controlling the gpt-selection marker,and the S4-promoter controlling the glu-plasminogen gene) is isolatedfrom a low melting point agarose gel and purified. This cassette isligated with arms of vaccinia virus vdTK DNA cut with NotI. Packagingand plaque purification are described in Example 3. A virus having thecorrect structure for the inserted plasminogen-gene cassette isdesignated vN2gpt-GPg. This virus is used for expression of plasminogenin CV-1 cells as described for an analogous vaccinia virus constructedby marker rescue techniques. Secreted glu-Pg in cell culturesupernatants was detected at a level of about 1.5 μg/10⁶ cells after 24hours of infection with a conventionally constructed vaccinia virusunder standard conditions for cultivation of vaccinia virus vectors forexpression of foreign proteins in cell culture. The glu-plasminogen inthe cell culture supernatant was detectable only in the presence of aprotease inhibitor (50 μg/ml of aprotinin).

Construction of a vaccinia virus (vLPg1) expressing humanlys-plasminogen: A sequence encoding lys-plasminogen was prepared bydeletion of the 231 bp coding region for the first 77 amino acids (Glu1to Lys77) of plasminogen from the complete plasminogen cDNA as shown inFIG. 5.3. This sequence was inserted into the gene expression cassetteof a plasmid (pN2gpt-S4) having a selectable marker gene (E. coli gpt),resulting in the plasmid designated pN2gpt-LPg (FIG. 5.3).

In this plasmid, the pre-sequence (coding for the signal peptide thatmediates secretion) is directly fused with the first nucleotide oflysine residue 78 in plasminogen. The novel signal peptide cleavage sitecreated by the fusion is similar to many known signal cleavage sites.See, for instance, von Heinje, Eur. J. Biochem. 133:17-21 (1983).

In addition, an NcoI site was introduced at the site of the initiationcodon of the Pg cDNA to facilitate cloning into the single NcoI site ofthe plasmid pN2gpt-S4 and to achieve the optimal context of the promoterand the Pg-coding region. To facilitate excision of Pg cDNA with NcoI,one of two internal NcoI sites (NcoI (2); FIG. 5.3) was deleted from thePg cDNA, as follows.

The plasmid phPlas6 was transferred into E. coli strain NM522 andsingle-stranded DNA was prepared by superinfection with the helper phageM13K07. The first round of mutagenesis was done with twooligonucleotides, oNco1 and oNco2, using the single-stranded phPlas6 DNAas a template with a commercially available mutagenesis kit (Amersham,Inc.). The oligonucleotide Nco1 converts two A-residues upstream of theplasminogen start codon into two C-residues, resulting in an NcoI sitearound the start codon without changing the coding region of theplasminogen pre-sequence. The oligonucleotide oNco2 converts a T into aC residue within the internal NcoI site (NcoI(2)) of the Pg cDNA,producing a silent mutation that inactivates this NcoI site.

The coding region for amino acids 1-77 of plasminogen was deleted bysecond loop-out mutagenesis step using 42-base oligonucleotide oNco3.All mutations were confirmed by sequencing and restriction analysis.

The plasmid having the three mutations, phLplas, was linearized withSmaI and partially digested with NcoI. The 2.2 kb NcoI-SmaI fragment wasisolated and inserted into plasmid pN2gpt-S4 that had been cut with NcoIand SmaI. The resulting plasmid was designated pN2gpt-LPg.

Due to the many modifications of the plasminogen cDNA in pN2gpt-LPg, thefull sequence of the NcoI-SmaI fragment of pLplas including 20 bases ofthe S4 promoter and 20 bases of the downstream plasmid region ofpN2gpt-S4 is shown in SEQ ID NO:18. The plasminogen cDNA sequence wasmodified as follows. The former two A-residues at positions #19 and #20(bases #53 and 54 in ID HSPMGR) were changed into two C-residues,resulting in an NcoI site; base #21 this listing (#55 in ID HSPMGR) isthe A-residue of the plasminogen start codon; base #2220 (base #2485 inID HSPMGR) is the T-residue of the stop codon; base #111 in ID HSPMGR(base #77 this listing) was joined with base #343 in ID HSPMGR (base #78this listing) resulting in the deletion of the sequence coding for the“activation peptide”; the T-residue #926 (base #1191 in ID HSPMGR) waschanged into a C residue (conservative exchange) resulting in thedisappearance of an internal NcoI site.

To transfer the lys-plasminogen gene cassette into a vaccinia virusgenome, the NotI fragment of pN2gpt-LPg containing the gene expressioncassette comprised of two promoter-gene combinations (the P7.5promoter-gpt gene and the S4 promoter-lys-plasminogen gene) is ligatedwith NotI cleaved vaccinia virus vdTK vector DNA and packaged asdescribed in Example 7. An isolate having the proper structure for theinserted gene cassette, designated vN2gpt-LPg, is used for expression oflys-plasminogen in CV-1 cells under conditions used previously for aconventionally constructed recombinant under standard conditions forcultivation of vaccinia virus expression vectors for production ofproteins in cell culture. Secreted lys-Pg in cell culture supernatantswas detected at a level of about 1.0-2.0 μg/10⁶ cells after 24 hours ofinfection with the conventional recombinant. The lys-plasminogen in thecell culture supernatant was stable without addition of a proteaseinhibitor.

Construction of a vaccinia virus (vgp160-1) for expressing humanimmunodeficiency virus glycoprotein 160 (HIV gp160): The complete openreading frame of HIV gp160 is obtained on a 2.5 kb EcoRV fragmentcontaining excised from replicative form (RF) DNA of an M13 phagemPEenv. Fuerst et al., Mol. Cell. Biol. 7:2538-2544 (1987). Thisfragment is inserted into the plasmid pN2gpt-S4 as outlined in FIG. 5.4.In the resulting plasmid, pN2gpt-gp160, the gp160 gene is controlled bythe synthetic vaccinia virus promoter S4.

The sequence of HIV gp160 has been published by Ratner et al., Nature313:277-284 (1985). The sequence of clone BH8 is accessible in the EMBOData Library (GenBank) under the Identifier (ID) HIVH3BH8. Therefore,the gp160 sequence is not included in SEQ ID NO:19, but the joiningregion of the S4 promoter and an EcoRV HIV-gp160 fragment including theinitiation codon of gp160 gene (base #28 this listing; base 226 in IDHIVH3BH8) is shown. The EcoRV HIV-gp160 fragment stems from the M13phage (replicative form) mPEenv described by Fuerst et al., Mol. Cell.Biol. 7:2538-2544 (1987). The sequence continues with the stop codon(base #31 this listing; base #2779 in ID HIVH3BH8) and one half of thedownstream EcoRV site. This sequence is followed by 20 bases of themultiple cloning site of plasmid pN2gpt-S4. The first base (T) of thislisting corresponds to base #3368, the last base (G), to #5973 in thesequence of pN2gpt-gp160.

To transfer the HIV gp160 gene-expression cassette into a vaccinia virusgenome, the NotI fragment containing both gene-promoter combinations(the P7.5 promoter-gpt selection marker and the S4 promoter-gp160 gene)is ligated with NotI-cleaved DNA of the vaccinia virus vector vdTK andpackaged as described in Example 7. An isolate having the correctstructure of insertion of the cassette, designated vN2gpt-gp160, is usedfor expression of gp160 in African green monkey (Vero) cells underconditions used previously for a conventionally constructed recombinant.Barrett et al., AIDS RESEARCH AND HUMAN RETROVIRUSES 6:159-171 (1989).

Construction of a vaccinia virus vector providing for screening formodified viruses carrying insertions by coinsertion of a lacZ gene: Todemonstrate the screening for insertion by coinsertion of an E. colilacZ gene in combination with the direct cloning approach, the plasmidpTZgpt-S3AlacZ provides a useful model construct (FIG. 5.5). The plasmidpTZ19R (Pharmacia, Inc.) was cut with Pvull, and the large 2.5 kb vectorfragment was prepared and ligated with NotI linkers (Boehringer, Inc.).The resulting plasmid, pTZ-N, has a deletion of the multiple cloningsite that is located within the sequences of the alpha complementationpeptide in the pT219R plasmid. Therefore, possible recombination eventsbetween the lacZ gene to be inserted into PTZ-N and the sequences of thealpha complementation peptide are excluded.

To construct a gene expression cassette for direct molecular cloning,the 1.2 kb NotI fragment, containing the gpt gene cassette and the S3Apromoter, is excised from pN2gpt-S3A (Example 4) and inserted into pTZ-Nresulting in the plasmid pTZgpt-S3A. The 3.0 kb EcoRI lacZ fragment,excised from plasmid pTKgpt-F1sβ described by Falkner and Moss (1988),is inserted into the single EcoRI site of pTZgpt-S3A. The resultingplasmid designated pTZgpt-S3AlacZ.

The 4.4 kb NotI fragment of this plasmid, consisting of the two markergenes (E. coli gpt and lacZ), is ligated with NotI cleaved DNA of thevirus vdTK (Example 4). The ligation and packaging conditions aredescribed in Example 3. The estimated yield of modified viruses in thecase of gpt-selection is described in Example 3.

An additional vaccinia virus vector was constructed as follows. Theplasmids pTZS4-lacZa and pTZS4-lacZb provided useful model constructs(FIG. 5.6). Plasmid PTZ-N was constructed as above. The gene expressioncassette, the 1.2 kb NotI fragment containing the gpt-gene cassette andthe S4 promoter was excised from pN2gpt-S4 (Example 4) and inserted intoPTZ-N resulting in the plasmid pTZgpt-S4. A 3.3 kb SmaI-StuI lacZfragment was excised from plasmid placZN*, which was constructed bydigesting the plasmid pFP-Zsart (European Patent Application No. 91 114300.-6, Recombinant Fowlpox Virus) with NotI and ligating pFP-Zsart withthe oligonucleotide P-NotI⁻ (5′-GGCCAT-3′). This 3.3 kb SmaI-StuI lacZfragment was inserted into the single SmaI site of pTZgpt-S4. Theresulting plasmids were designated pTZS4-lacZa and pTZS4-lacZb. The 4.5kb NotI fragment of this plasmid was ligated with the NotI cleaved DNAof the virus vdTK and packaged as described above.

The combination of lacZ and gpt-selection in a single cloning stepoffers no advantage because all gpt-positive plaques will contain thelacZ gene. However, for the construction of viruses having insertions indifferent sites, a second screening procedure is desirable. The markerof first choice is the gpt marker, but lacZ screening offers analternative method for detection of inserts, for instance, when thetarget viral genome already contains a copy of a selectable marker suchas the E. coli gpt gene.

For such screening, two ml of 1/10, 1/100 and 1/1000 dilutions of crudevirus stocks prepared after packaging (see Example 3) is plated on 30large (diameter of 8.5 cm) petri dishes (10 petri dishes per dilution).The blue plaque assay is done according to standard procedures.Chakrabarti et al., Mol. Cell. Biol. 5:3403-3409 (1985).

EXAMPLE 6 Construction of a vaccinia virus vector (vS4) with adirectional master cloning site under transcriptional control of astrong late vaccinia virus promoter

The present example describes a vaccinia virus cloning vector (vS4) thatis designed for direct molecular insertion of a complete open readingframe into a master cloning site that is functionally linked to avaccinia virus promoter. Accordingly, use of this vector according tomethods of the present invention enables insertion of genes directlyinto a poxvirus vector without separate construction of an insertionplasmid, as required in conventional construction of recombinantpoxviruses by intracellular recombination. This vector also obviates theneed for separate construction of a gene expression cassette fortransfer into a vaccinia virus vector by direct molecular insertion, asdescribed hereinabove.

The master cloning site of vector S4 is located in the geneticallystable central region of the vaccinia virus genome and is comprised ofseveral cleavage sites that are unique in the vector, thus permittingdirectional insertion. The S4 promoter immediately upstream of themaster cloning site is a strong synthetic variant of a late vacciniavirus promoter. This expression vector is suitable for direct cloningand expression of large open reading frames which include a translationstart codon, as illustrated here by a cDNA encoding a human bloodprotein, the von Willebrand factor (vWF).

Construction of the vaccinia virus vector vS4: An adaptor containing thesynthetic vaccinia virus promoter S4 is inserted into the vaccinia virusvector vdTK (Example 4, FIGS. 4.1A-C) at the unique NotI site (FIG.6.1). Insertion of the selected adaptor oligonucleotides inactivates theupstream NotI site while the downstream NotI site remains functional asa unique cloning site.

More particularly, DNA (1 μg) of the vector vdTK (Example 4, FIGS.4.1A-C) is cleaved with NotI and ligated with (0.5 μg) annealedoligonucleotides P-artP(11) (SEQ ID NO:38) and P-artP(12) (SEQ IDNO:39). The ligation mix is packaged and plaques are identified asdescribed in Example 3. Plaques are subjected to PCR screening asdescribed (Example 4, Identification of the virus vdTK by PCRscreening). An isolate having the insert in the correct orientation isdesignated vS4.

Insertion of the von Willebrand factor cDNA into vS4: Plasmid pvWFcontains the complete von Willebrand factor cDNA flanked by NotI sites.The sequence of human vWF has been published by Bonthron, D. et al.,Nucl. Acids Res. 14:7125-7128 (1986). The sequence is accessible in theEMBO Data Library under the Identifier (ID) HSVWFR1. SEQ ID NO:20 showsthe junction in the virus genome of vvWF of the viral S4 promoter andthe 5′-untranslated region of the present vWF cDNA in the plasmid pvWFup to the translational start codon (base #249 in this listing; base#100 in ID HSVWFR1). The coding region of vWF was omitted in the instantsequence listing. The sequence continues with the stop codon (base #252;base #8539 in ID HSVWFR1) and the 3′-untranslated sequence up to theNotI site (base #304) and twenty bases of overlap with the 3′-region ofthe viral genome of vvWF.

The vWF CDNA fragment is released with NotI, isolated and ligated withvS4 vector DNA that has been cleaved with NotI and treated withphosphatase, as illustrated in FIG. 6.2.

One μg of ligated DNA is packaged as described in Example 7. Plaques arepicked and analyzed by PCR screening. The first primer for the PCRreaction is oligonucleotide odTK2 which is located about 300 bp upstreamof the tk-gene; the reverse primer ovWF1 is located in the vWF geneabout 50 bp downstream of the initiation codon. PCR amplification occursonly when the vWF insert is in the correct orientation relative to theS4 promoter in the vector. PCR-positive plaques are identified andanalyzed further. Alternatively, if the yield of desired modified virusis low, on the order of 0.1 to 0.01%, then they may be identified by insitu plaque hybridization methods adapted from those known in the art.See, for instance, Villareal and Berg, Science 196:183-185 (1977).

A virus clone having the cDNA insert by PCR or hybridization and furthershowing the expected restriction pattern with PvuII is designated vvWF.Such vectors may be tested for expression of von Willebrand factor asdescribed for other human proteins in Example 5, modified as appropriateaccording to genetic engineering principles well known by one skilled inthis art.

EXAMPLE 7 Heterologous packaging of orthopox (vaccinia) virus genomicDNA by an avipox (fowlpox) helper virus and simultaneous selection formodified virus in host cells of a species in which the helper viruscannot replicate

Example 3 describes packaging of modified vaccinia virus DNA withfowlpox helper virus in avian cells and subsequent isolation of progenyvirus plaques in mammalian (CV-1) cells in which the avipox helper viruscannot replicate. The present example illustrates packaging of vacciniavirus DNA by fowlpox directly in CV-1 cells, thereby permittingsimultaneous packaging and host range selection for packaged virus.Besides eliminating helper virus from the initial stock of progeny, thisprocedure circumvents the tedious requirement for producing primarycultures of chicken embryo fibroblasts for each packaging experiment.Instead, continuous. mammalian cell lines that are commonly used forvaccinia virus replication also can be used for packaging vaccinia viruswith fowlpox helper virus.

It is known that fowlpox virus (FPV) replicates completely only in aviancells; no viable progeny virus is obtained from infected mammaliancells. The precise point in the life cycle of FPV at which replicationis aborted in mammalian cells is not known. However, FPV is known toproduce viral proteins in mammalian cells and even to induce protectiveimmunity in mammals when used as a live vaccine. Taylor et al., Vaccine6:497-503 (1988). Nevertheless, FPV has not been shown previously tohave a capacity for packaging heterologous poxvirus genomic DNA,particularly directly engineered vaccinia virus DNA.

In an initial experiment, CV-1 cells (5×10⁶) were infected with onepfu/cell of fowlpox virus (strain HP1.441) and incubated for one hour.Subsequently, a calcium-phosphate precipitate (one ml containing one μgof vaccinia virus wildtype DNA) was transfected into the infected cells.After 15 min at room temperature, 10 ml of medium (DMEM, 10% fetal calfserum) were added. The cells were incubated for four hours, and themedium was changed. The cells were then incubated for six days, and acrude virus stock was prepared. The progeny virus were titered on CV-1cells. Typical vaccinia plaques were visible after two days.

The dependence of packaging efficiency on the amount of genomic viralDNA was determined over a range of DNA amounts from 0.1 to 10 μg per5×10⁶ CV-1 cells. See FIG. 7.1. Amounts of DNA in excess of 1 μg (e.g.,10 μg) produced a coarse calcium-phosphate precipitate that reduced theefficiency of transfection in terms of pfu/μg of input DNA. FIG. 7.1.

The dependence of the packaged vaccinia virus yield on the incubationtime for packaging was analyzed using a constant amount of vacciniavirus wildtype DNA (1 μg) and a constant amount of FPV helper virus (1pfu/cell) under the conditions described above for the initialexperiment in this example except that the medium added 15 minutes aftertransfection was changed after four hours, and the cells were thenincubated for an additional 1 to 5 days before preparing a crude virusstock (total volume of 2 ml). Virus stock from control cells infectedwith FPV only and incubated for 5 days produced no visible plaques. Thisexperiment was repeated three times and a typical outcome is shown inTable 4, below.

TABLE 4 Effect of incubation time on yield of vaccinia virus from DNApackaging by fowlpox helper virus in mammalian (CV-1) cells. IncubationTime Titer (hours) (pfu/ml) 24 1.0 × 10² 48 4.6 × 10⁴ 72 5.0 × 10⁵ 965.6 × 10⁶ 120 2.1 × 10⁷

The titer of packaged vaccinia virus, detected by plaque assay onmammalian (CV-1) cells, rose continually from about 10² pfu/ml at 24hours to about 2×10⁷ after 120 hours. Incubation times in the range of48 to 72 hours produced convenient levels of packaged vaccinia virus(between 10⁴ and 10⁶ pfu/ml) and, therefore, are suitable for routinepackaging of vaccinia virus DNA by fowlpox virus in mammalian cells.

Vaccinia DNA can be packaged in mammalian cells abortively infected withfowlpox virus. It was shown previously that fowlpox virus can alsoinfect mammalian cells, but the viral life cycle is not completed inthese non-typic host cells. Depending on the cell type, viral growthstops either in the early or in the late stage and viable fowlpox virusis not formed. Taylor et al. (1988). These findings prompted aninvestigation into packaging vaccinia DNA in a continuous mammalian cellline. Confluent monolayers of CV-1 cells were infected with 0.05 pfu percell of the FPV strain HP1.441 and then transfected with a ligationmixture consisting of NotI-cleaved vaccinia virus DNA and a gpt genecassette having NotI flanking sites. More particularly, vaccinia DNA (1μg) was digested with NotI and ligated with indicated amounts of insertDNA (P7.5 gpt gene cassette). The unique NotI site in vaccinia virus islocated in an intergenic region in the HindIII F fragment. Goebel etal., Virology 179:247 (1990). After incubation for three days the cellswere harvested and the crude virus stock was titered on CV-1 cells inthe presence (+MPA) and in the absence (−MPA) of gpt-selective medium.The outcome is summarized in Table 5.

TABLE 5 Titers after abortive packaging titers (pfu × 10⁻²/6 × 10⁶cells) chimeras expt. # insert (ng) −MPA* +MPA (%) 1. 200 17.2 1.6 9.32. 200 42.5 5.1 12.3 3. 400 64.0 3.8 5.9 4. 400 26.8 3.8 14.2 5. 210.0*MPA, mycophenolic acid.

The most important result was that fowlpox virus could package themodified vaccinia DNA in a cell type that prevents its own growth.Moreover, the yield of chimeric plaques was in the range of 5-10%. Thiscompares favorably with the classical in vivo recombination technique,in which usually about 0.1% of the total plaques are recombinants.Ligation of the vector arms alone (Table 5, experiment #5) resulted in ahigher titer compared to ligation experiments 1-4 with insert, probablydue to lack of contaminants present in the agarose-purified insertmolecules.

Some of the isolated viruses were plaque-purified and furthercharacterized. They showed the typical HindIII restriction patterns ofvaccinia virus and, in addition, foreign gene bands characteristic forthe two possible orientations of the single insert. With insertion intothe NotI site, no viruses with multiple inserts were observed.

Heterologous packaged chimeric vaccinia viruses do not cross hybridizewith fowlpox virus. In order to study the effects of heterologouspacking by FPV on the structure of chimeric vaccinia viruses, DNAs ofisolates F13.4, F12.5, F13.2, F13.2 and F12.4, together with those offour purified isolates from the NotI cloning experiment and the fowlpoxvirus controls, were digested with HindIII, and the resulting fragmentswere separated by electrophoresis and analyzed by Southern hybridizationwith a fowlpox virus probe prepared from sucrose gradient-purifiedvirions. No cross hybridization of the vaccinia viruses with FPV DNA wasobserved.

EXAMPLE 8 Homologous packaging of engineered vaccinia virus genomic DNAby a vaccinia virus host range mutant (vdhr) that is unable to replicatein a human cell line

The present example illustrates construction and utilization of a helperpoxvirus comprised of deletions that limit its host range, particularlythe ability to replicate in certain human cell lines. Therefore,modified vaccinia virus free of helper virus can be prepared bypackaging of vector DNA with this mutant helper virus and isolatingclones of the engineered virus by infecting appropriate human cells.

This mutant helper virus is derived from host range mutants of vacciniavirus which are unable to replicate in a variety of human cells andwhich display altered cytopathic effects on many other cells that arepermissive for infection by wildtype vaccinia virus. See, for example,Drillien et al., Virology 111:488-499 (1981). In particular, the genomeof this helper virus comprises mutations of two host range genes whichtogether prevent it from replicating in human (MRC 5) cells in whichonly vaccinia virus genomes having at least one intact host range genecan replicate.

Construction of the host range mutant vaccinia virus vdhr: The genomiclocation and DNA sequence of one vaccinia virus gene required forreplication in human cells has been described by Gillard et al., Proc.Natl. Acad. Sci. USA 83:5573-5577 (1986). Recently, this gene has beendesignated KiL. Goebel et al. (1990). A second vaccinia virus host rangegene has been mapped. Perkus et al., J. Virology 63:3829-2836 (1990).This second gene, designated C7L by Goebel et al. (1990), lies in aregion encompassing parts of the HindIII C and HindIII N fragments. Thisregion is deleted in the vaccinia virus WR6/2 strain. Moss et al., J.Virol. 40:387-395 (1981). Strain WR-6/2 therefore lacks the C7L hostrange gene.

The helper virus vdhr lacking both the K1L and C7L host range genes isconstructed from the C7L-negative strain WR-6/2 by marker rescue with amodified EcoRI K fragment from which the K1L host range gene is deleted.See FIG. 8.1. This modified EcoRI K fragment comprises a selectivemarker gene (the E. coli gpt gene) to facilitate selection for modifiedWR-6/2 genomes comprising the modified EcoRI K fragment usingintracellular marker rescue. Sam and Dumbell (1981). A conditionallethal mutant which lacks the ability to grow on human cell lines hasalso been described. Perkus et al. (1989).

More particularly, the 5.2 kb EcoRI K fragment of vaccinia viruswildtype DNA is subcloned into the plasmid pFP-tk18i. The resultingplasmid is designated pFP-EcoK1. The vaccinia virus host range gene K1L,see Gillard et al. (1986), is deleted and simultaneously a unique NotIsite is introduced by loopout mutagenesis using the oligonucleotideP-hr(3) (SEQ ID NO:42). The resulting plasmid is designated pEcoK-dhr.

The plasmid pFP-tk18i was constructed by modification of the plasmidpFP-tk-10.4. See Falkner et al. application, Example 3 at 8. PlasmidpFP-tk10.4 was digested with NcoI and ligated with an adaptor consistingof annealed nucleotides P-NcoI(1) and P-NcoI (2), resulting in theintroduction of a multiple cloning site into the single NcoI site of theFPV tk-gene with the restriction endonuclease cleavage sites EcoRI, NotIand

The sequence of vaccinia virus has been published by Goebel et al.,Virology 179:247-266 (1990). It is accessible in the EMBO Data Library(GenBank) under the Accession Number M35027. The sequence of thevaccinia virus host range gene K1L has been published by Gillard et al.,Proc. Natl. Acad. Sci. USA 83:5573-5577 (1986), and is accessible in theEMBO Data Library (GenBank) under the Identifier (ID) PXVACMHC.Therefore, the coding sequence of the K1L gene is not included in SEQ IDNO:21. In pEcoK-dhr the K1L gene is deleted and replaced by a NotI site.The joining region between the PXVACMHC sequence and the NotI siteinsert is shown (bases #1-20 of this listing correspond to bases #72-91in ID PXVACMHC). The coding region of K1L was deleted and replaced by aNotI site followed by two G residues (bases #21-30 in the sequencelisting). The sequence continues with 20 bp flanking region (bases#31-50 this listing; bases #944-963 in ID PXVACMHC).

In a further step pEcoK-dhr is linearized with NotI and ligated with a1.1 kb P7.5-gpt gene cassette derived from plasmid pN2-gpta (Example 4)by NotI digestion. The resulting plasmid pdhr-gpt is used generate thehelper virus vdhr.

The NotI cassette (comprising the P7.5 promoter-gpt gene cassette)inserted into pEcoK-dhr and twenty bases of the 5′ and 3′ flankingregions are shown for pdhr-gpt in SEQ ID NO:22. The flanking region(bases #1-20 this listing) correspond to bases #72-91 in ID PXVACMHC(see SEQ ID NO:21 for pEcoK-dhr). The inserted DNA sequence starts atposition 21 (the first “G” of a NotI site) and ends at position 1189(the last “C” residue of a NotI site). The A-residue of thetranslational initiation codon of the gpt gene corresponds to position#548. The T-residue of the translational stop codon of the gpt genecorresponds to position number #1004. The sequence continues with 20bases of flanking region (bases #1192-1209 this listing; bases #944-961in ID PXVACMHC). The two “G” residues #1190 and 1191 in this listing,correspond to position 29 and 30 of pEcoK-dhr.

To transfer the Eco K fragment into vaccinia virus, the plasmid istransfected into primary chicken embryo fibroblasts cells infected withthe vaccinia virus deletion mutant WR-6/2. Modified viruses are selectedas gpt-positive (using mycophenolic acid). A gpt-positive isplaque-purified three times in CEF cells and designated vdhr.

Characterization of the vdhr helper virus: The structure of gpt-positivevaccinia virus vdhr is analyzed by Southern blot analysis and host rangetests. The vdhr virus is capable of forming plaques on chicken embryofibroblasts and two monkey cell lines (BSC40 and Vero) but is defectivefor replication in the human cell line MRC-5.

Packaging of engineered vaccinia virus DNA using the host range mutantvdhr as a helper virus: A construct for expression of a cDNA encodinghuman prothrombin demonstrates the utility of this approach. The productfrom a ligation mixture described in Example 5, FIG. 5.1, is transfectedinto chicken embryo fibroblasts infected with vdhr as a helper virus.After 2 days the cells are harvested and a crude virus stock isprepared. Packaged virus is assayed for plaque formation on human (MRC5) cells in which the desired vaccinia virus replicates but the mutantvdhr helper virus does not.

After three days the cells are stained with neutral red and plaques areselected for further analysis by Southern blot. Modified vaccinia virusclones having the desired structure are identified. Viruses which haveundergone recombination with the highly homologous helper virus are alsoexpected.

EXAMPLE 9 Construction of novel chimeric vaccinia viruses encoding HIVgp160 (vP2-gp160_(MN)A, vP2-gp160_(MN)B and vselP-gp160_(MN)) andexpression of recombinant gp160_(MN) in Vero cells

The present example illustrates construction by direct molecular cloningof a vaccinia virus recombinant for large scale production of gp160 ofthe HIV-1_(MN) isolate. Production of the gp160 of the HIV-1_(IIIB)isolate described by Ratner et al., Nature 313:277-284 (1985), using aconventionally constructed vaccinia virus expression vector, has beendescribed by Barrett et al., AIDS Res. Human Retroviruses 5:159-171(1989). The HIV-1_(IIIB) isolate, however, is a rare HIV variant.Efforts at developing vaccines based on HIV envelope proteins shouldinclude more representative HIV-1 isolates such as the MN-isolate. Gurgoet al., Virology 164:531-536 (1988); Carrow et al., Aids Res. HumanRetroviruses 7:831-839 (1991). Accordingly, the present vaccinia virusvectors were constructed via direct molecular cloning to express thegp160 protein of the HIV_(MN) isolate.

Construction of the plasmid pP2-gp160_(MN) and of the chimeric virusesvP2-gpt160_(MN)A and vP2-gp160_(MN)B: The strategy of inserting thegp160-gene into vaccinia virus involved (i) modifying the gp160-gene byremoving the large 5′-untranslated region (5′-UTR) and introducing asuitable cloning site upstream of the start codon, (ii) cloning themodified gp160-gene downstream of the strong late fowlpox virusP2-promoter (European Patent Application No. 91 114 300.-6, Aug. 26,1991) and (iii) inserting a blunt-ended fragment consisting of theP2-gp160 and P7.5-gpt gene cassettes into the single restrictionendonuclease cleavage sites of appropriate viral host strains, e.g. intothe SmaI site of the host vaccinia strain vdTK (Example 4), the SmaI orthe NotI sites of the vaccinia strain WR 6/2 of Moss et al., J. Virol.40:387 (1981), or the vaccinia wild-type strain WR.

For these purposes, a new SmaI site was introduced into the plasmidpN2gpt-S4 (Example 4), resulting in the plasmid pS2gpt-S4 (FIG. 9.1, SEQID NO:62). Subsequently the S4-promoter was exchanged by the P2-promoterresulting in the plasmid pS2gpt-P2 (SEQ ID NO:63). This plasmid allowsthe cloning of complete open reading frame (orf's) but can also be usedto clone incomplete orfs lacking their own start codon; the start codonis provided, for instance, when cloning into the single NcoI site(CCATGG) of this plasmid. Construction of the plasmids and viruses isdescribed in further detail below. For the modification of thegp160-gene, a PCR-generated proximal fragment was exchanged leading to agp160-gene cassette with a minimal 5′-UTR. This cassette is present inthe final construct, the plasmid pP2-gp160_(MN) (FIG. 9.1, SEQ IDNO:69). Additional characteristics of the plasmid are shown in table 6below.

TABLE 6 pP2qp160mn (SEQ ID NO:69) Location Description 1-3529 pS2gpt-P2sequences 2396-2851 rcCDS of E. coli gpt gene 2851 T of rc initiationcodon TAC of the gpt gene 2395 A of the rc stop codon of TTA 3081-3323rc of vaccinia P7.5 promoter 3358-3526 P2 promoter sequence according toEP application Avipox “intergenic region”. 3534-6001 CDS of the HIV-1strain MN gp160 sequence (EMBL ID REHIVMNC) 3534 A of the initiationcodon ATG of the gp160MN 6102 T of the stop codon TAA of the gp160MN6173-6926 pS2gpt-P2 sequences

The plasmids were constructed as follows:

pS2gpt-S4: The plasmid pN2gpt-S4 (Example 4) was digested with XbaI andligated with a SmaI-adaptor (SEQ ID NO:43: 5′-CTAGCCCGGG-3′)inactivating the XbaI and creating a SmaI site. The resulting plasmidwas designated pS2gpt-S4 (SEQ ID NO:62).

Additional characteristics of this plasmid are shown in the table 7below.

TABLE 7 pS2gpt-S4 (SEQ ID NO:62) Location Description 1-2226 pN2gpt-S4sequences of SEQ ID NO:14. Position 1 corresponds to the firstnucleotide G ‘5-TGGCACTTT TCGGGGAAAT-3′ (bases 2-20 of SEQ ID NO:62).2227-2236 SmaI-adaptor 5′-CTAGCCCGGG-3′ (SEQ ID NO:43). 2396-2851 rcCDSof E. coli gpt gene 2851 T of rc initiation codon TAC of the gpt gene2395 A of the rc stop codon of TTA 3081-3323 rc of vaccinia P7.5promoter 3358-3451 S4-promoter of SEQ. ID#14 (oligonucleotide P- artP(9) see p. 120) 2237-4145 pN2gpt-S4 sequences of SEQ. ID No. 14

pS2gpt-P2: The S4-promoter segment of plasmid pS2gpt-S4 was removed bycleavage with PstI and HpaI and replaced with a 172 bp PstI-HpaIP2-promoter segment. This promoter segment was generated by PCR with theplasmid pTZgpt-P2a (Falkner et al., European Patent Application No. 91114 300.-6, Aug. 26, 1991) as the template and the oligonucleotides P-P25′(1) and P-P2 3′(1) as the primers. The PCR-product was cut with PstIand HpaI and ligated the PstI and HpaI-cut large fragment of pS2gpt-S4.The sequence of P-P2 5′(1) (SEQ ID NO:44) is: 5′-GTACGTACGG CTGCAGTTGTTAGAGCTTGG TATAGCGGAC AACTAAG-3′; the sequence of P-P2 3′(1) (SEQ IDNO:45) is: 5′-TCTGACTGAC GTTAACGATT TATAGGCTAT AAAAAATAGT ATTTTCTACT-3′.The correct sequence of the PCR fragment was confirmed by sequencing ofthe final plasmid, designated pS2gptP2 (SEQ ID NO:63). The sequenceprimers used were P-SM(2) (SEQ ID NO:46), 5′-GTC TTG AGT ATT GGT ATTAC-3′ and P-SM(3) (SEQ ID NO:47), 5′-CGA AAC TAT CAA AAC GCT TTA TG-3′.Additional characteristics of the plasmid pS2gpt-P2 are shown in table 8below.

TABLE 8 pS2gpt-P2 (SEQ ID NO:63) Location Description 1-3357 ps2gpt-S4sequences 2396-2851 rcCDS of E. coli gpt gene 2851 T of rc initiationcodon TAC of the gpt gene 2395 A of the rc stop codon of TTA 3081-3323rc of vaccinia P7.5 promoter 3358-3526 P2 promoter sequence according toEP application Avipox “intergenic region”. 3527-4277 pS2gpt-S4 sequences

pMNevn2: The plasmid pMNenvl was provided by R. Gallo (National CancerInstitute, Bethesda, Md.). It contains the gp160-gene of the HIVMN-strain cloned as a 3.1 kb EcoRI-PvuII fragment in the vector pSP72(Promega, Inc.). The 0.6kb EcoRI-Asp718 fragment of pMNenvl was replacedwith a 0.13 kb EcoRI-Asp718 fragment, removing large parts of the5′-untranslated region of the gp160-gene. This 0.13 kb fragment wasgenerated by PCR using the plasmid pMNenvl as the template and theoligonucleotides P-MN(1) and P-MN(2) as the primers. The forward primerP-MN(1) introduced, in addition, a StuI site 1 bp upstream of the startcodon. The sequence of P-MN(1) (SEQ ID NO:48) is 5′-AGCTAGCTGAATTCAGGCCT CATGAGAGTG AAGGGGATCA GGAGGAATTA TCA-3′; the sequence ofP-MN(2) (SEQ ID NO:49) is 5′-CATCTGATGC ACAAAATAGA GTGGTGGTTG-3′. Theresulting plasmid was designated pMNenv2. To exclude mutations the PCRgenerated fragment in this plasmid was sequenced with the primers P-Seq(2) (SEQ ID NO:50) 5′-CTG TGG GTA CAC AGG CTT GTG TGG CCC-3′ andP-Seq(3) (SEQ ID NO:51) 5′-CAA TTT TTC TGT AGC ACT ACA GAT C-3′.

pP2-gp160MN: The 2.7 kb StuI-PvuII fragment, containing the MNgp160-gene, isolated from the plasmid pMNenv2 was inserted into the HpaIsite of pS2gpt-P2 resulting in the plasmid pP2-gp160MN (SEQ ID NO:69).

The chimeric viruses vP2-gp160_(MN)A and vP2-gp160_(MN)B wereconstructed as follows. The SmaI-fragment consisting of the P2-gpl60 andP7.5-gpt-gene cassettes was inserted by direct molecular cloning intothe single SmaI site of the host vaccinia strain vdTK (Example 4)resulting in the chimeric viruses vP2-gp160_(MN)A and vP2-gp160_(MN)B(FIG. 9.2). In particular, the vaccinia virus vdTK of Example 4 was cutat its single SmaI site and ligated with the 4.0 kb SmaI fragment thatcontains the P7.5-gpt-gene and the P2-gp160 gene cassettes.Correspondingly, the vaccinia strain WR6/2 was cut at its single SmaI(NotI) site and ligated with the 4.0 kb SmaI (NotI) fragment thatcontains the P7.5-gpt gene and the P2-gp160 gene cassettes. The cloningprocedures were carried out as described in Example 1. In the virusvP2-gp160_(MN)A, the gp160 gene is transcribed in the same direction asthe genes clustered around the viral thymidine kinase gene; in the virusvP2-gp160_(MN)B, the gp160 gene is transcribed in the reverse direction.Since gene position effects can influence expression levels in vacciniaconstructs, the SmaI (NotI)-fragment consisting of the P2-gp160 andP7.5-gpt gene cassettes was also inserted into the SmaI (NotI) site ofthe WR 6/2 strain. The in vivo packaging was done as described inExample 3.

Structure of the chimeric viruses. To confirm the theoretical structuresof the chimeric viruses (FIG. 9.3), Southern blot analyses are carriedout. DNA's of the purified viruses are cleaved with PstI and resultingfragments are separated on an agarose gel, transferred to anitrocellulose membrane and hybridized to a vaccinia thymidine kinase(tk) gene and a gp160-gene probe. With the tk gene probe, in the case ofvP2-gp160_(MN)A, the predicted 6.9 and the 14.3 kb fragments arevisible, and for vP2-gp160_(MN)B, the predicted 8.7 and 12.5 kbfragments are visible. With the gp160 probe (pMNenv1), the predicted14.3 kb of vP2-gp160_(MN)A and 8.7 kb fragment of vP2-gp160_(MN)B arevisible, confirming the integration of the foreign gene cassettes in twodifferent orientations.

Expression studies with the chimeric viruses vP2-gp160_(MN)A andvP2-gp160_(MN)B. Vero cells are chosen for expression studies. Growth ofcells, infection with the chimeric viruses and purification of therecombinant gp160 protein are carried out as described by Barrett etal., supra.

Western blot analysis of gp160: The Western blot analysis are doneessentially as described by Towbin et al., Proc. Natl. Acad. Sci. USA83:6672-6676 (1979). The first antibody is a mouse monoclonal anti-HIVgp120 antibody (Du Pont, Inc. #NEA9305) used at a 1:500 dilution. Thesecond antibody is a goat-anti-mouse IgG (H+L) coupled with alkalinephosphatase (BioRad, Inc., #170-6520) used at a 1:1000 dilution. Thereagents (BCIP and NBT) and staining protocols are from Promega, Inc.

Construction of the plasmid pselP-gp160MN and of the chimeric virusvselP-gp160_(MN). The synthetic early/late promoter selP (SEQ ID NO:70)which is one of the strongest known vaccinia virus promoters, was usedin this example to express the gp160-gene of the HIV-1 MN strain. SeeEuropean Patent Application No. 91 114 300.-6. First, the plasmidpselP-gpt-L2 was constructed (FIG. 9.4). This plasmid includes theselP-promoter followed by a multiple cloning site for the insertion offoreign genes, as either complete or incomplete open reading frames, andtranslational stop codons in all reading frames followed by the vacciniavirus early transcription stop signal, TTTTTNT. Rohrmann et al., Cell46:1029-1035 (1986). The P7.5 qpt gene cassette is located adjacent tothe promoter and serves as a dominant selection marker. Falkner et al.,J. Virol. 62:1849-1854 (1988). The selP-promoter/marker gene cassettesare flanked by restriction endonuclease cleavage sites that are uniquein the vaccinia virus genome (SfiI, NotI, RsrII) and can also be excisedas blunt ended fragments (for instance, by cleavage with HpaI andSnaBI). To be able to insert the gp160 gene into pselP-gpt-L2, an NcoIsite was introduced around the translational start codon. This mutationresults in the substitution of the amino acid arginine (AGA) withalanine (GCC). This mutation in the second amino acid of the signalpeptide is not likely to interfere with efficient expression of thegp160-gene. The cloning procedure and the sequence around the wildtypeand the modified gp160-gene is outlined in FIG. 9.5A. FIG. 9.5B showssequences around translational start codons of wild-type (SEQ ID NO:73)and modified gp160 genes (SEQ ID NO:75). To introduce the mutation intothe gp160-gene, a PCR-generated proximal fragment was exchanged. Theconstruction of the plasmids is described in more detail below.

pL2: For the construction of pL2, the 0.6 kb XbaI-ClaI fragment of theplasmid pTM3, see Moss et al., Nature 348:91 (1990), was substituted byan XbaI-ClaI adaptor fragment consisting of the annealedoligonucleotides o-542 (SEQ ID NO:52) 5′-CGA TTA CGT AGT TAA CGC GGC CGCGGC CTA GCC GGC CAT AAA AAT-3′ and o-544 (SEQ ID NO:53) 5′-CTA GAT TTTTAT GGC CGG CTA GGC CGC GGC CGC GTT AAC TAC GTA AT-3′. The intermediateplasmid resulting from this cloning step was called pL1. The 0.84 kbAatII-SphI fragment (parts of noncoding gpt-sequences) were substitutedby the AatII-SphI adaptor fragment consisting of the annealedoligonucleotides o-541 (SEQ ID NO:54: 5′-CTT TTT CTG CGG CCG CGG ATA TGGCCC GGT CCG GTT AAC TAC GTA GAC GT-3′) and o-543 (SEQ ID NO:55: 5′-CTACGT AGT TAA CCG GAC CGG GCC

ATA TAG GCC GCG GCC GCA GAA AAA GCA TG-3′). The resulting plasmid wascalled pL2.

pTZ-L2: The XbaI-SphI fragment (consisting of theT7-promoter-EMC-T7-terminator segment, the multiple cloning site and theP7.5-gpt gene cassette) was treated with Klenow-polymerase and insertedbetween the PvuII sites of the plasmid pTZ19R (Pharmacia, Inc.). Theresulting plasmid was called pTZ-L2 (SEQ ID NO:64). Additional featuresof this plasmid are shown in table 9 below.

TABLE 9 pTZ-L2 (SEQ ID NO:64) Location Description 1-55 pTZ19R sequences(Pharmacia) 56-108 Linker I in rc orientation (5TNT, NotI, SfiI, RsrII,HpaI, SnaBI, AatII 110-860 E. coli gpt sequences in rc orientation. Thegpt open reading frame starts with a rc TAC start codon at position 860and ends with a rc ATT stop codon at pos 403 861-1338 Vaccinia Virusp7.5 promoter sequences in rc orientation 1339-1344 HpaI site betweenbacteriophage T7 terminator and Vaccinia Virus p7.5 promoter 1345-1488Bacteriophage T7 terminator sequences in rc orientation. See Dunn &Studier, J. Mol. Biol. 166: 477-535 (1983) 1489-1558 Multiple cloningsite in rc orientation (SalI, translation stop codons for all three openreading frames, StuI, XhoI, PstI, BamHI, SpeI, SaoI, SmaI, EcoRI, NcoI)1559-2131 sequences from the Encephalomyocarditis Virus (EMC- Virus)5′untranslated region ( ) in rc orientation 2132-2187 Bacteriophage T7promoter sequences in rc orientation ( ) 2190-2242 Linker II in rcorientation (SnaBI, HpaI, NotI, SfiI, 5TNT) 2243-4701 pTZ19R sequences(Pharmacia)

PTZselP-L2 and pselP-gpt-L2: The 0.6 kb ClaI-NcoI fragment (theT7-promoter-EMC-sequence) was replaced with a synthetic promoterfragment consisting of the annealed oligonucleotides o-selPI (SEQ IDNO:56: 5′-CGA TAA AAA TTG AAA TTT TAT TTT TTT TTT TTG GAA TAT AAA TAAGGC CTC-3′; 51 mer) and o-selPII (SEQ ID NO:57: 5′-CAT GGA GGC CTT ATTTAT ATT CCA AAA AAA AAA AAT AAA ATT TCA ATT TTT AT 3′). The resultingintermediate plasmid pTZselP-L2 still contains the T7-terminator and aHpaI site, that were removed in the following cloning step therebyinserting a vaccinia early transcription stop signal and reducing thesize of the P7.5 promoter fragment from 0.28 to 0.18 kb. The 239bpSaII-NdeI fragment was substituted by the SaII-NdeI adaptor consistingof the annealed oligonucleotides o-830 (SEQ ID NO:58: 5′-TCG ACT TTT TATCA-3′) and o-857 (SEQ ID NO:59: 5′-TAT GAT AAA AAC-3′). The resultingplasmid was called pselP-gpt-L2 (SEQ ID NO:65). Additional features ofthis construct are shown in table 10 below.

TABLE 10 pselP-gpt-L2 (SEQ ID NO:65) Location Description 1-55 pTZ19Rsequences (Pharmacia) 56-108 Linker I in rc orientation (5TNT, NotI,SfiI, RsrII, HpaI, SnaBI, AatII) 110-860 E. coli gpt sequences in rcorientation. The gpt open reading frame starts with a rc TAC start codonat position 860 and ends with a rc ATT stop codon at position 403861-1245 Vaccinia Virus p7.5 promoter sequences in rc orientationstarting with the p7.5 internal NdeI site at position 1241 1246-1258Vaccinia Virus early transcription stop signal in rc orientation flankedby a NdeI site (position 1245) and a SalI site (position 1253) 1259-1322Multiple cloning site in rc orientation (SalI, translation stop codonsfor all three reading frames, StuI, XhoI, PstI, BamHI, SpeI, SacI, SmaI,EcoRI, NcoI) 1323-1374 Vaccinia Virus synthetic early late promoter inrc orientation flanked by a NcoI site at position 1317 and a ClaI siteat position 1370 1375-1414 Linker II in rc orientation (SnaBI, HpaI,NotI, SfiI, 5TNT) 1415-3878 pTZ19R Sequences (Pharmacia)

pselP-gp160MN: The 3.1 kb env gene containing the EcoRI-PvuII fragmentof pMNenvI was inserted into the EcoRI and StuI cut plasmid pselP-gpt-L2resulting in the intermediate plasmid pselP-gpl60.1. The 0.8 kbNcoI-NsiI fragment of pselP-gp160 was substituted by a PCR-generated0.31 kb NcoI-NsiI fragment resulting in the final plasmidpselP-gp160_(MN) (SEQ ID NO:66). Additional features of this plasmid areshown in table 11 below.

TABLE 11 pselP-gp160MN (SEQ ID NO:66) Location Description 1-55 pTZ19Rsequences (Pharmacia) 56-108 Linker I in rc orientation (5TNT, SfiI,RsrII, HpaI, SnaBI, AatII) 110-860 E. coli gpt sequences in rcorientation. The gpt open reading frame starts at position 860 with a rcTAC start codon and ends at position 403 with a rc ATT stop codon861-1245 Vaccinia Virus p7.5 promoter sequences in rc orientationstarting with the p7.5 internal NdeI site at position 1241 1246-1258Vaccinia Virus early transcription stop signal in rc orientation(position 1245-1252) flanked by a NdeI site at position 1241 and a SalIsite at position 1253. 1259-3916 HIV-1 MN env gene in rc orientation.The ORF starts at position 3916 with a rc TAC start codon and ends atposition 1348 with a rc ATT stop codon 3917-3970 Vaccinia Virussynthetic early late promoter in rc orientation flanked by a NcoI site(position 3913) and a ClaI site (position 3966) 3971-4015 Linker II inrc orientation (SnaBI, HpaI, NotI, SfiI, 5TNT) 4016-6474 pTZ19Rsequences (Pharmacia)

The primers used for the PCR reaction were o-NcoI (40mer) SEQ ID NO:60:5′-GAG CAG AAG ACA GTG GCC ATG GCC GTG AAG GGG ATC AGG A-3′, and o-NsiI(30mer) SEQ ID NO:61: 5′-CAT AAA CTG ATT ATA TCC TCA TGC ATC TGT-3′. Forfurther cloning the PCR product was cleaved with NcoI and NsiI.

Chimeric viruses vselP-gp160_(MN)A vselP-gp160_(MN)B: The HpaI-fragmentconsisting of the selP-gp160 and P7.5gpt gene cassettes is inserted bydirect molecular cloning (FIG. 9.6) into the single SmaI site of thevaccinia strain WR6/2which is a highly attenuated vaccinia virus strain.Moss et al., J. Virol. 40:387-395 (1981); Buller et al., Nature317:813-815 (1985). The vaccinia virus strain WR6/2 is cut at its singleSmaI site and ligated with the 4.0 kb HpaI fragment that contains theP7.5-gpt gene and the selP-gp160-gene cassettes. The cloning proceduresare carried out as described in Example 1.

The resulting chimeric viruses, vselP-gp160_(MN)A and vselP-gp160_(MN)B,are purified and further characterized. In the virus vselP-gp160_(MN)A,the gp160 gene is transcribed in the same direction (left to right) asthe genes clustered around the insertion site [the A51R open readingframe. Goebel et al., Virology 179:247-266 (1990). In the virusvselP-gp160_(MN)B, the gp160-gene is transcribed in the reversedirection. The in vivo packaging is done as described in Example 3.

Structure of the chimeric viruses: To confirm the theoretical structuresof the chimeric viruses (FIG. 9.7), Southern blot analyses are carriedout. The DNA of the purified viruses was cleaved with SalI and fragmentsare separated on an agarose gel, transferred to a nitrocellulosemembrane and hybridized to vaccinia SalF-fragment probe (pTZ-SalF) and agp160 gene probe (pMNenv1). With the SalF-fragment probe, forvselP-gp160_(MN)A the predicted 6.8 and 10.7 kb fragments are visible;and for vselP-gp160_(MN)B, the predicted 3.5 and 13.7 fragments arevisible. With the gp160 probe, the same fragments are seen, but the 10.7kb fragment in vselP-gp160^(MN)A and the 3.5 kb fragment invselP-gp160_(MN)B give less intense signals, because only about 400 bpof each total fragment is homologous to the probe.

Since direct cloning also results in integration of tandem multimerstructures, the DNA of the viruses is also digested with XbaI which doesnot cut the inserted DNA. The XbaI wild-type fragment is 447bp in size.Integration of one copy of the 3.8 kb sized insert results in a fragmentof 4.3 kb. In multimeric structures the size of the 4.3 kb fragmentincreases in increments of 3.8 kb.

Expression studies with the chimeric viruses vselP-gp160_(MN)A andvselP-gp160_(MN)B: Vero cells are used for expression studies. Growth ofcells, infections with the chimeric viruses and purification of therecombinant gp160 protein are carried out as described by Barrett etal., supra.

EXAMPLE 10 Construction of novel chimeric vaccinia viruses encodinghuman protein S (vProtS) and expression of recombinant protein S

This example illustrates the construction of recombinant protein Sexpressed by chimeric vaccinia virus. Human protein S is a 70 kDaglycoprotein involved in the regulation of blood coagulation. DiScipioet al., Biochemistry, 18:890-904 (1979). The cDNA and the genomic DNA ofProtein S have been cloned and characterized. Lundwall et al., Proc.Natl. Acad. Sci. USA 83:6716 (1986); Hoskins et al., Proc. Natl. Acad.Sci. USA 84:349 (1987); Edenbrandt et al., Biochemistry, 29:7861 (1990);Schmidel et al., Biochemistry 29:7845 (1990)

Human protein S, normally synthesized as a 70 kDa protein in liver andendothelial cells, see DiScipio et al., supra, has been expressed inpermanent cell lines derived from human 293 and hamster AV12-664 cells(adenovirus transformed cell lines) at levels of up to 7 μg/10⁶cells/day, see Grinnell et al., Blood 76:2546 (1990), or in mouse C127cell/papilloma virus system at similar expression levels, see Malm etal., Eur. J. Biochem. 187:737 (1990). The protein derived from thelatter cells was larger than plasma-derived protein S probably due toaberrant glycosylation.

The present expression of protein S uses a double gene cassetteconsisting of the complementary DNA for the human blood factor protein Sand the gpt gene, each controlled by a vaccinia promoter. This wascloned into the unique NotI site and packaged in fowlpox helpervirus-infected mammalian cells. Human protein S was expressed ininfected Vero cells in levels of 4-6 μg per 10⁶ cells.

For the cell screening, for optimal protein S expression by the chimericvaccinia virus, five different host cell lines were used, WI 38 (humanembryonal lung fibroblast), CV-1 and Vero (monkey kidney cells), Changliver and SK Hep1. Protein S was indistinguishable from plasma-derivedprotein S by several criteria: the recombinant material derived from theinfected cells of this cell line showed the same electrophoreticmigration patterns and the same chromatographic elution profiles asplasma-derived protein S. This indicates that the correctpost-translational modification of this complex glycoprotein hasoccurred. The methods are described in detail below.

Construction of the plasmid pN2-gptaProtS. Single-stranded DNA preparedfrom the plasmid pBluescript-ProtS, comprising the cDNA coding for humanprotein S (provided by R. T. A. McGillivray) was used to mutagenize theregion around the translational start codon of the protein S codingregion into an NcoI site (CCATGG). The mutagenic primer, oProtS1 (SEQ IDNO:68), has the sequence 5′-ACC CAG GAC CGC CAT GGC GAA GCG CGC-3′; themutagenesis was carried out as described in the mutagenesis protocol(Amersham, Inc.). The signal peptide is mutated, with the second aminoacid changed from Arg to Ala (FIG. 10.1B and SEQ ID NOS. 78 and 80) Thisintroduces the NcoI site required for further cloning and brings the ATGstart codon into an optimal context for translation. This may improvethe secretion of protein S.

The protein S cDNA was subsequently excised as an NcoI-NotI fragment andinserted into the vaccinia insertion plasmid pTKgpt-selP (Falkner etal., supra) a plasmid providing a strong synthetic vaccinia promoter.The promoter-protein S gene cassette was then excised as a BglII-NotIfragment and inserted into the plasmid pN2-gpta (Example 1) resulting inpN2-gptaProtS (see FIG. 10.1A; SEQ ID NO:67). Additional features ofthis construct are shown in table 12 below.

TABLE 12 pN2-gptaProtS (SEQ ID NO:67) Location Description 1-2217Bluescript II SK- sequences (Stratagene) 2218-2225 NotI site 1 2226-4938ProtS sequences in rc orientation. The open reading frame starts atposition 4938 with a rc TAC start codon and ends at position 2910 with arc ATT stop codon 4939-4992 Vaccinia Virus synthetic early late promoterin rc orientation flanked by a NcoI site (position 4935) and a fusedBglII/BamHI site (position 4987-4992). The NcoI site harbors the Prot Src start codon TAC 4993-5493 Vaccinia Virus p7.5 promoter sequences5494-6127 E. coli gpt sequences. The ORF starts at position 5494 withATG start codon and ends at position 5950 with a TAA stop codon.6228-6235 NotI site 2 6236-6811 Bluescript II SK- sequences (Stratagene)

In this plasmid, the gpt-gene controlled by the vaccinia virus P7.5promoter and the protein S cDNA, is transcribed divergently and flankedby NotI sites.

Insertion of the cDNA for human protein S into the single NotI site ofvaccinia virus to form vProtS. The NotI-fragment consisting of the gptgene and protein S gene cassettes was ligated with the vaccinia vectorarms and transfected into FPV infected mammalian CV-1 cells. Onlypackaged vaccinia virus multiplied under these conditions. Moreparticularly, vaccinia wild-type DNA of the WR strain (1 μg) was cutwith NotI, the enzyme was heat-inactivated for 30 min at 65° C. Thevector was ligated overnight with 1 μg of the 3.8 kb gpt/Protein S genecassette (excised as a NotI fragment out of the plasmid pN2gpta-ProtS)in 30 μl using 15 units of T4 DNA ligase.

The crude virus stocks prepared after five days of incubation weretitrated in the presence and in the absence of mycophenolic acid (MPA).This procedure distinguished chimeric from back-ligated wild-type virus.With MPA 4×10⁴ and without the drug 6×10⁵ pfu/10⁶ host cells wereobtained. About 6-7% of the viral plaques were chimeric viruses. Ten ofthe gpt-positive isolates were plaque-purified twice, grown to smallcrude stocks and were used to infect CV-1 cells. Total DNA was prepared,cut with the restriction enzymes SacI and NotI and subjected to Southernblot analysis (FIGS. 10.2A-C). The SacI digest, hybridized with thecloned SacI-I fragment (plasmid pTZ-SacI; Example 4), allowed thedetermination of the orientation of the inserted DNA because SacI cutsthe inserts asymmetrically. In all ten isolates the inserts were in the‘a’-orientation (fragments of 6.3 and 4.6 kb; see FIGS. 10.2A and10.2C), indicating that this configuration is strongly preferred. TheNotI fragments were hybridized with the protein S probe. In this casethe 3.8 kb NotI gene cassette was released (FIG. 10.2B).

Expression of human protein S by a chimeric vaccinia virus. Crude stockswere grown from gpt-positive chimeric viruses and used for infection ofvarious mammalian cell lines. Monolayers of 5×10⁶ cells were infectedwith 0.1 pfu/cell in the presence of serum free medium (DMEM)supplemented with 50 μg/ml vitamin K and incubated for 72 hours.Supernatants were collected and protein S antigen was determined usingan ELISA test kit from Boehringer Mannheim, FRG (Kit Nr. 1360264).Amounts of protein S synthesized are given in Table 13 in milli-units (1U corresponds to 25 μg of protein S).

Alternatively, 10 μl of supernatant from Vero cells were analyzed in aWestern Blot analysis using 50 ng of human plasma-derived protein S as astandard and a mouse polyclonal serum specific for “hu Prot S” (Axell)(FIG. 10.3). Blots were stained using an alkaline phosphatase conjugatedgoat anti-mouse polyclonal serum (Dakopatts) and NBT/BCIP as asubstrate.

Purification of recombinant protein S from cell culture supernatants wasperformed as described by Grinnell et al. (1990).

TABLE 13 Cell line ATCC# mU huProtS per 10⁶ cells SK Hepl (HTB52) 750Vero (CCL 81) 127 Chang Liver (CCL 13) 135 CV-1 (CCL 70) 450 WI 38 (CCL75) 440

EXAMPLE 11 Construction of novel chimeric vaccinia viruses encodinghuman factor IX and expression of recombinant factor IX

A double gene cassette consisting of the complementary DNA for the humanblood factor IX and the gpt gene, each controlled by a vacciniapromoter, was cloned into the unique NotI site of the vaccinia virus WRgenome and packaged in fowlpox helper virus-infected mammalian cells.Human factor IX was expressed in several cell types.

Human clotting factor IX is a 56 kDa glycoprotein involved in theregulation of blood coagulation. This clotting factor undergoes complexpost-translational modifications: vitamin K dependent carboxylation ofthe first 12 glutamic residues, glycosylation, 3-hydroxylation of anaspartic acid and amino terminal protein processing. Davie, E. W., “TheBlood Coagulation Factors: Their cDNAs, Genes and Expression”,HEMOSTATIS AND THROMBOSIS, Colman et al., eds., J. B. Lippincott Co.(1987). Hemophilia B, an X chromosome-linked bleeding disorder, iscaused by mutation of factor IX. Patients with hemophilia are currentlytreated by substitution with plasma-derived factor IX.

The cDNA and the genomic DNA of factor IX (“FIX”) have been cloned andcharacterized and FIX has been expressed in permanent cell lines. Busbyet al., Nature 316:271 (1985); Kaufman et al., J. Biol. Chem. 261:9622(1986); Balland et al., Eur. J. Biochem. 172:565 (1922); Lin et al., J.Biol. Chem. 265:144 (1990). Expression of factor IX in vacciniarecombinants has also been described. de la Salle, et al., Nature316:268 (1985).

Construction of plasmids—pN2gpta-FIX: The FIX cDNA (kindly provided byR. T. A. MacGillivray) was cut from pBluescript-FIX with EcoRI andligated with the EcoRI linearized plasmid pTM3. Moss et al., Nature348:91 (1990) Single strand DNA was isolated from a recombinant plasmidwhich contained the FIX insert in the correct orientation and a NcoIsite (CCATGG) was introduced around the FIX ATG start codon byoligonucleotide mediated site directed mutagenesis using oligonucleotideoFIX.1 (SEQ ID NO:71: 5′-TCA TGT TCA CGS GCT CCA TGG CCG CGG CCG CACC-3′) and a commercial mutagenesis kit (Amersham, Inc.; kit No. PPN1523). Vector and FIX NcoI sites were fused, insert DNA was isolated byNcoI and NotI digestion and ligated with the NcoI/NotI cut vectorpTKgpt-selP. Falkner et al., supra The promoter/FIX cassette was cut outfrom this plasmid with BglII and NotI and ligated with the BamHI/NotIlinearized vector pN2-gpta (Example 1). From this construct a NotIcassette containing the FIX cDNA (under the control of the selPpromoter) and the gpt gene (under the control of the vaccinia P7.5promoter) was isolated and used for in vitro molecular cloning andpackaging as described in Example 10. Additional characteristics of thisplasmid are shown in table 14 below.

TABLE 14 pN2gpta-FIX (SEQ ID NO:72) Location Description 1-2217Bluescript II SK-sequences (Stratagene) 2218-2225 NotI site 1 2226-3659FIX sequence in rc orientation. The open reading frame starts atposition 3659 with a rc TAC start codon and ends at position 2276 with arc ATT stop codon 3660-3713 Vaccinia Virus synthetic early late promoterin rc orientation flanked by a NcoI site (position 3656 and fusedBglII/BalII site (position 3708-3713). The NcoI site harbors the FIX rcstart codon TAC 3714-4214 Vaccinia Virus p7.5 promoter sequences4215-4848 E. coli gpt sequences. The ORF starts at position 4215 with anATG start codon and ends at position 4671 with a TAA stop codon.4849-4856 NotI site 2 4857-5532 Bluescript II SK-sequences (Stratagene)

Insertion of the cDNA for human Factor IX into the single NotI site ofvaccinia virus. Prior to insertion of the factor IX cDNA into vacciniavirus, this cDNA was inserted into the plasmid pN2-gpta resulting in theplasmid pN2gpta-FIX (FIG. 11.1A, SEQ ID NO:72). To obtain the optimalsequence context between the synthetic vaccinia promoter and the factorIX coding region, the 5′ untranslated region of factor IX was deleted byintroduction of a novel NcoI site at the start codon of factor IX andfusion of this NcoI site with the NcoI site provided by the promoter.This mutation resulted in a mutated signal peptide (FIG. 11.1B, SEQ IDNOS 81-84).

In the wildtype factor IX the second amino acid of the signal peptide isa glutamine residue while in pN2gpta-FIX the second amino acid is aglutamic acid residue.

The NotI fragment consisting of the gpt-gene and factor IX genecassettes was ligated with the vaccinia vector arms and transfected intoFPV infected mammalian CV-1 cells. Only packaged vaccinia virusmultiplied under these conditions. The crude virus stocks prepared afterfive days of incubation were titrated in the presence and in the absenceof mycophenolic acid (MPA). This procedure distinguished chimeric fromback ligated wild-type virus. With MPA 5×10⁴ and without the drug 5×10⁶pfu/10⁶ host cells were obtained. In this example, about 1% of the viralplaques were chimeric viruses. Ten of the gpt-positive isolates wereplaque-purified twice, grown to small crude stocks and were used toinfect CV-1 cells. Total DNA was prepared from eight cell culturesinfected with the respective viral isolates, digested with therestriction enzymes SfuI, Ndel and NotI and subjected to Southern blotanalysis.

The SfuI digest, hybridized with the factor IX probe, allowed thedetermination of the orientation of the inserted DNA because SfuI cutsthe inserts asymmetrically. In all eight isolates the inserts were inthe ‘a’-orientation (fragments of 6.3 and 4.6 kb; see FIG. 11.2A),indicating that this configuration is strongly preferred. The NdeI(NotI) fragments were also hybridized with the factor IX probe. In thiscase a fragment of 6.6 kb (the 3.8 kb NotI gene cassette) was released,proving the predicted structure.

Expression Of Human Factor IX. Crude stocks were grown from eight singleplaque isolates and used for infection of various mammalian cell lines.5×10⁶ cells in a 10 cm petri dish were infected with a moi of 0.1pfu/cell in the presence of serum free medium (DMEM) and 50 μg/mlvitamin K. Infected cells were incubated for 72 hours until cellsstarted to detach from the bottom of the petri dish. Supernatants werecollected, cell fragments were removed by centrifugation and FIX amountswere determined using an ELISA test kit from Boehringer Mannheim, FRG(Kit Nr. 1360299). Amounts of FIX antigen and of factor IX activitiesare given in Table 15.

Alternatively, 10 μl of supernatant from Vero cells were analyzed in aWestern Blot analysis using 50 ng of human plasma derived huFIX as astandard and a mouse polyclonal serum specific for huFIX (Axell). Blotswere stained using an alkaline phosphatase conjugated goat anti-mousepolyclonal serum (Dakopatts) and NBT/BCIP as a substrate. As shown inFIG. 11.3, the recombinant material migrated as a broad band similar tothe plasma-derived factor IX standard. Clotting assays of the partiallypurified Vero cell derived factor IX showed that about 50% of thematerial was active factor IX. The virus isolate #5, designated VFIX#5,was grown to large scale and used for further experiments.

As in the case of the protein S chimeric viruses (Example 10), thefactor IX expressing chimeras had inserts in one preferred orientation.

The protein of transcription of the gene of interest (factor IX andprotein S) was from right to left, i.e., the same direction as the genesclustered around the NotI site. It seems therefore, that stronglytranscribed units have to be aligned in the preferred transcriptionaldirection when cloned into the NotI cluster. Viruses with thisconfiguration of the insert are strongly preferred and show the bestgrowth characteristics. The direction of transcription of the secondgene cassette, the P7.5 gpt gene, was from the left to right. The P7.5promoter segment is therefore in an inverted repeat configurationrelative to the nearby endogenous gene coding for the 7.5 kDa protein,i.e. the expected stable configuration is preferred. Since no chimeraswith the reverse orientation were found, the ‘b’-orientation is probablyunstable. Insertion of the above mentioned gene cassettes in the‘b’-orientation by in vivo recombination would have failed, leading tothe misinterpretation that the NotI intergenic region is essential forviral growth. This situation illustrates one of the advantages of thedirect cloning approach: only ‘allowed’ are structures are formed.

By insertion of simple small gene cassettes, both orientations andmultimers were obtained (Example 1) while insertion of complex genecassettes (divergently transcribed double gene cassettes with homologiesto internal genes such as the P7.5 promoter segment) preferredstructures were formed.

The cell screening for optimal factor IX expression showed thatinfection of CV-1 and SK Hep1 cells resulted in the highest antigenlevels. The material from CV-1 cells had the highest clotting activities(table 15), indicating that this cell line possesses effectivepost-translational modification systems. Factor IX has been expressedpreviously in the conventional vaccinia expression system using the P7.5promoter and HepG2 and BHK cells (de la Salle et al., 1985). Cell lineswith better growth characteristics, like Vero and CV-1 cells, have beenshown to produce higher levels of expression with the instant viruses,due to improved promoters and methods. In addition, deletion of the5′-untranslated region of the factor IX cDNA and the modification of thesignal peptide seems to have positive effects on secretion andexpression levels.

TABLE 15 Factor IX Expression in Different Cell Lines activity ratiocell line ATCC# antigen (mU/10⁶ cells)* % SK Hepl (HTB52) 810 183 22.5Vero (CCL81) 500 282 56.4 Chang Liver (CCL13) 190 100 52.6 CV-1 (CCL70)850 1290 51.8 RK13 (CCL37) 300 460 53.3 *1 unit corresponds to 5 μg FIXper ml human plasma

EXAMPLE 12 Construction of the chimeric fowlpox virus f-envIIIB andexpression of recombinant HIVIIIB envelope proteins in chicken embryofibroblasts

The large scale production of gp160 in a vaccinia virus-Vero cellexpression system has been described recently. Barrett et al. (1989).Since vaccinia virus is still pathogenic to many vertebrates includingmammals and fowlpox virus is host restricted to avian species we havedeveloped an avipox based expression system. See U.S. Ser. No.07/734,741 and CIP thereof. Chimeric fowlpox viruses have now beenconstructed by direct molecular cloning to express the envelope gene ofthe HIV-1 IIIB isolate. Ratner et al. (1985). In this recombinant virusthe env gene is controlled by a strong synthetic late promoter. For theproduction of envelope glycoproteins, the chimeric fowlpox virus is usedto infect chicken embryo aggregate cell cultures. Mundt et al.,PCT/WO91/09937.

Construction and structure of the chimeric fowlpox virus f-envIIIB. Forconstruction of f-envIIIB (FIG. 12.1) a double gene cassette consistingof the P7.5-promoter/gpt gene and the S4-promoter/gp160 gene wereexcised as a NotI-fragment out of the plasmid pN2gpt-gp160 (Example 5).This cassette was ligated with NotI-cleaved genomic DNA of the fowlpoxvirus f-TK2a (Example 2) and chimeric virus was isolated as described inMaterials and Methods. Total DNA from chicken embryo fibroblastsinfected with twelve different plaques was digested with SspI andfurther analyzed by Southern blot analysis and hybridization with anisolated gp160 fragment as a probe (FIG. 12.2A). The predicted fragmentsof 3.7, 1.0 and 0.8 kb were found in 11 cases indicating that the gp160gene had been integrated in the ‘b’-orientation (FIG. 12.2B). One viralisolate, f-LF2e, did not hybridize to the gp160 probe.

The fact that one preferred orientation of the insert exists, points tothe possibility that the ‘b’-orientation virus has growth advantagesover the ‘a’-orientation, the ‘a’-orientation may even be unstable.Letting the viral vector choose the best orientation may be consideredas an advantage of the direct cloning approach.

Expression studies with the chimeric virus f-envIIIB. Expression studieswere done in chicken embryo fibroblasts (CEF). Confluent monolayers ofCEFs were infected with 0.1 pfu per cell of the different viral crudestocks, grown for five days. Total cellular proteins were separated on10% polyacrylamide gels, transferred onto nitrocellulose membranes andfurther processed as described in Materials and Methods. A Western blotanalysis showing the expression of gp160, gp120 and gp41 is shown inFIGS. 12.3 and 12.4. All viral isolates, except f-LF2e, inducedexpression of the env glycoproteins. The virus f-LF2e was also negativein the Southern blot analysis and therefore does not carry the gp160gene sequences.

Construction of f-envIIIB. Two micrograms of DNA of host virus vectorf-Tk2a (Example 2) were cut with NotI and ligated with 500 nanograms ofthe gene cassette consisting of the P7.5-promoter/gpt gene and theS4-promoter/gp160 gene. The ligation was carried out in a volume of 20μl and 5U of ligase for four days at 12° C. The ligation mixture wastransfected into 6×10⁶ CEFs infected with 0.5 pfu per cell of HP2, afowlpox isolate obtained by plaque-purification of HP1.441. After anincubation period of five days a crude stock was prepared (final volume1 ml) which was amplified. The crude stock was titrated on CEFs insix-well plates and grown for 5 days under gpt-selection (25 μg/mlmycophenolic acid, 125 ug xanthine). Cells on which the minimal dilutionresulted in a visible cytopathic effect, were harvested and amplifiedtwice according the same protocol. The crude stock obtained from thesecond amplification from the second amplification was titered on CEFsin the presence of gpt-selection and 12 single plaques (f-LF2a-1) werepicked.

Western blot analysis of gp160. The Western blot analysis were doneessentially as described by Towbin et al., supra. For gp160/gp120detection, the first antibody was a mouse monoclonal anti-HIV gp120antibody (Du Pont, Inc. # NEA9305 used at a 1:500 dilution. For the gp41detection the human anti-HIV-gp41 3D6 Mab (provided by H. Katinger,Universitat fur Bodenkultur, Inst. fur Angewandte Mikrobiologie) wasused at a 1:500 dilution. The second antibody was a goat-anti-mouse IgG(H+L) coupled with alkaline phosphate (BioRad, Inc. #170-6520) used at a1:1000 dilution. The reagents (BCIP and NBT) and staining protocols arefrom Promega, Inc.

EXAMPLE 13 Construction of the chimeric vaccinia virus vRMN6b1 andexpression of recombinant gp160MN in Vero cells

In Example 9, the construction of chimeras expressing gpl6O, under thecontrol of the fowlpox virus (FPV) P2 promoter, described in EPA91.114.300.6, is set forth. This promoter is a strong late promoter.Since it is desirable not only to use the vaccinia gpl6OMN constructsfor production purposes, but also as live vaccines, new constructs thatexpress gp16OMN early and late in the viral live cycle were made. TheFPV P2 promoter was synthetically modified such that an earlytranscription promoting sequence was inserted downstream of the latepromoter region. This hybrid promoter was designated “Sep.” The HIVgp16OMN sequence was cloned downstream of this promoter.

Unexpectedly high expression levels of gp16OMN were obtained with thesespecific constructs. The Sep-controlled gp16OMN is expressed at similaror higher levels as in the T7-double infection system. Fuerst et al.(1987). The T7-double infection system has a major drawback, however,requiring two different viruses to express a single antigen. The virusvRMN6b1 is used to produce gp16OMN and supplants the need for thegpl60/T7-double infection system.

Construction of the plasmid pSep-ST2 and of the chimeric virus vRMN6b1:The plasmid pSep-ST2 contains the HIV-1 gp16OMN sequences controlled bythe strong semi-synthetic poxvirus promoter Sep and a selection cassetteconsisting of the P7.5 promoter gpt-gene. Falkner and Moss (1988). Thisplasmid was assembled from two plasmids, obtained from Dr. M. Reitz(NCI, Bethesda, Md.), designated pMNenvl and pMN-ST2, and with theplasmid pSep(l). FIG. 13.1. The construction of the plasmid psep(l) andthe structure of the Sep promoter is outlined in FIG. 13.2 (SEQ ID NOS87-89). The “late” region of this promoter is based on the ‘P2’-promoterdescribed in U.S. Ser. No. 07/935,313, which was provided with an“early” component by ligation with specific oligonucleotides. FIG. 13.2.

To construct the virus vRMN6b1, the double gene cassette, excised as aNotI fragment out of the plasmid pSep-ST2 and consisting of the P7.5-gptselection marker and the Sep regulated gp16OMN gene, was inserteddirectly into the NotI site of the WR-WT strain.

Six gpt-positive viruses were plaque purified six times and screened forexpression of the env protein by Western blot analysis. Three of themdid not express gp16OMN (the viruses vRMNI.1, vRMN2.11, vRMN3.1) whilethe other three isolates (vRMN4.11, vRMN6b1, vRMN8.11) showed a strongsignal in the 160 kDa size range (FIG. 13.5). The virus vRMN6b1 wasfinally chosen on the basis of its high expression level for furthercharacterization.

Structure of the chimeric virus vRMN6b1: Direct molecular cloning ofinserts into a unique viral restriction site can result in differentgenomic structures. Scheiflinger et al. (1992). The most common ones arethe orientational isomers of the insert. Interestingly the insert of allsix gpt-positive viruses had the ‘b’-orientation, i.e., the direction oftranscription of the Sep-gpl6O cassette is from right to left (from thecentral part to the left terminus). To confirm the theoretical structureof the chimeric virus vRMN6b1 Southern blot analyses were carried out(FIGS. 13.3 and 13.4). The DNA's of the purified viruses vRMN6b1 andWR-WT (control) were cleaved with several restriction enzymes, separatedon an agarose gel, transferred to a nitrocellulose membrane andhybridized to a gpl6O gene probe (FIG. 13.3). An identical blot washybridized with a probe obtained by PCR amplification of the regionaround the NotI site (“the Not-region probe”) of the wild-type virusgenerated with the primers P-N(l) and P-N(2) (FIG. 13.4).

Using the gpl6O gene probe pMNenv1 and the Not-region probe, thepredicted fragments were visible. Some of the smaller fragments wereonly visible after longer exposure times (not shown). The predictedsizes of the different fragments are summarized in Table 16. As expectedthe WR-WT virus did not hybridize with the gpl6O probe.

The chimeric virus vRMN6b1 induces very high levels of gp160: Toestimate gpl6O expression levels with a known, efficient system, the T7bacteriophage polymerase/vaccinia hybrid system was used to generate acomparative Western blot analysis for the expression of gpl6O induced byvRMN6b1. Fuerst et al. (1987); Barrett et al. (1989). Confluentmonolayers of cells were infected with 0.1 pfu's of the respectiveviruses and, after 48 hours, total proteins were analyzed. The highestlevels were obtained with vRMN6b1 in CV-1 cells. FIG. 13.6, lane vRMN6b1CV-I′. Interestingly, expression levels in Vero cells were similar tothose obtained in the vaccinia virus phage T7 polymerase hybrid system.FIG. 13.6. This blot shows that a strong, early/late promoter can beoptimized for very high expression levels of gpl6O. With vRMN6b1 as avehicle only one virus is required for the production of gpl6O, ascompared to two viruses with the T7 hybrid system, thereby reducingeffort and cost of production of gpl6O.

Methods: The plasmids pMNenvl and pMN-ST2 were provided by M. Reitz(NCI, Bethesda, Md.). The construction of the plasmid pMNenv2 isdescribed above. Briefly, a 2.65 kb StuI/PvuII fragment derived from theplasmid pMNenv2, containing the HIV 1-MN env gene, was ligated with theSnaBI- linearized plasmid pSep(l). The construction of psep(l) isdescribed in the legend of FIG. 13.2. The resulting plasmid, containingthe insert in the proper orientation with respect to the “Sep-promoter”was designated pSep-gpl6Omn. In order to repair a point mutation locatedwithin the gpl6O-orf, a 1.9 kb NsiI/SalI fragment of pSep-gpl6Omn wasreplaced by an equivalent fragment derived from the plasmid pMN-ST2. Theresulting plasmid was designated pSep-ST2.

The semi-synthetic poxvirus promoter Sep was constructed by combinationof the late fowlpoxvirus promoter P2 with a synthetic early promotersequence. Briefly, the HpaI/NcoI digested vector pS2gpt-P2 (see U.S.Ser. No. 07/914,738) was ligated with the annealed oligonucleotidesP-Sep(3) and P-Sep(4). The sequence of the oligonucleotides was P-Sep(3)(SEQ ID NO:90) 5′:CTCGTAAAAA TTGAAAAACT ATTCTAATTT ATTGCACGGT CGCGA-3′;and P-Sep(4) (SEQ ID NO:91): 5′-CATGGTACGT ACCGTGCAAT AAATTAGAATAGTTTTTCAA TTTTTACGAG-3′. The resulting plasmid was designated pSep(l).

The viruses were digested with the restriction enzymes SalI, HindIII,PstI, NotI, XbaI and HpaI. The fragments were hybridized to the³²P-labeled gpl6O probe pMNenv1. The marker (m) consisted of phagelambda HindIII fragments and of phage phi X HaeIII fragments. The markersize is indicated in kilobasepairs.

The viruses were digested with the restriction enzymes SalI, HindIII,PstI, NotI, XbaI and HpaI. The fragments were hybridized to a³²P-labeled PCR probe (generated with the primers P-N(l) (SEQ ID NO:92),5′-GCTCCCGCAG GTACCGATGC AAATGGCCAC-3′, and P-N(2) (SEQ ID NO:93),5′-GGGGAGAGAT CGAAAGTGAA TTTGACATAGC-3′, and the a template consistingof WR-WT virus. The marker (m) consisted of phage lambda HindIIIfragments and of phage phi X HaeIII fragments; the marker size isindicated in kilobasepairs.

The Western blot analysis were done essentially as described by Towbinet al. (1979). The first antibody was the human monoclonal anti-HIV-gp41antibody 3D6 used at a 1:500 dilution. Grunow et al. (1988). The secondantibody was a goat anti-human IgG coupled with alkaline phosphatase(BioRad, Inc. #172-1004) used at a 1:1000 dilution. The reagents (BCIPand NBT) and staining protocols are from Promega, Inc.

TABLE 16 Sizes of genomic restriction endonuclease fragments of theviruses vRMN6bl and WR-WT theoretically hybridizing to the gpl60 and theNot-region probes (fragment sizes are given in kilo basepairs, kb).vRMN6b1 WR-WT Not- Not- enzyme gpl60 pr. region pr. gp160 pr. region pr.SalI 26.6 + 0.75 26.6 + 0.75 — 23.3 HindIII 12.8 + 1.3 + 1.2 12.8 + 2.3 — 13.5 PstI 5.9 20.9 + 5.9  — 22.8 Notl 4.0 145 + 45  — 145 + 45 XbaI5.7 5.7 —  1.6 Hpal 5.2 5.2 —  1.1

EXAMPLE 14 Construction of the chimeric vaccinia virus vgag (1) andexpression of recombinant HIV gag protein

Human immunodeficiency virus type 1 (HIV-1) contains an RNA genome thatencodes gag, pol, and env proteins, as well as additional regulatoryproteins. Ratner et al. (1985); Sanchez-Pescador et al. (1985). Theprimary gag translation product is a 55 kDa precursor, Pr55gag, that isnormally processed into the major core proteins p24, p17, and p15 byproteolysis. p15 is, in turn, cleaved into p7 and p6. Veronese et al.(1988). A myristic acid residue is present at the N-terminus of both p17and the gag precursor. Veronese et al. (1988); Mervis et al. (1988). Byanalogy to other retroviruses, the myristic acid likely is required fortransport of viral proteins to the plasma membrane. Rein et al. (1986).HIV gag and gag/pol proteins have been expressed in several expressionsystems, such as yeast, vaccinia system and baculovirus. Kramer et al.(1986); Walker et al. (1987); Flexner et al. (1988); Gowda et al.(1989); Karacostas et al. (1989); Shioda and Shibuta (1990); Hu et al.(1990); Madison et al. (1987); Gheysen et al. (1989). Expression of thegag precursor (Prs55gag) alone, without the HIV protease, leads to theformation of virus-like particles. Gheysen et al. (1989). They arelikely candidates for vaccine preparations either for subunit vaccinesor as components of live vaccines.

In this example, the efficient expression of the gag precursorcontrolled by the early late promoter Sep in a chimeric virus isdescribed. The virus may be used for the production of HIV-1 gagprotein. The chimeric virus has some unique properties such as delayedonset of cytopathic effects in Vero cells and an attenuated phenotype.The latter makes the chimeric virus especially useful as a safe andefficient vector for the production of gag proteins and gagpseudoparticles for vaccine and diagnostic use.

Construction of the plasmid pSep-gag and of the chimeric viruses vgag(1)and vgag(2): The HIV gag sequence was derived from the plasmid pMN-ST2provided by M. Reitz (NCI, Bethesda, Md.). It was subcloned from pMN-ST2into pBluescript IISK-, shown in FIG. 14.1, and designated pgagMN(S/H).To shorten the 5′-untranslated region of the gag sequence and tosubsequently introduce an NcoI site at the gag translation initiationcodon, the 150 bp ClaI-SacI fragment of pgagMN(S/H) was replaced by afragment annealed from the synthetic oligonucleotides P-gag(1) (SEQ IDNO:94) and P-gag(2) (SEQ ID NO:95). The gag ORF was inserted intopSep(l), resulting in psep-gag, from which the Sep-gag and P7.5-gptgenes can be excised as a 3.1 kb NotI fragment.

Chimeric viruses were constructed as described above. Three originalisolates (#1, #2 and #7) were further plaque-purified six times bygpt-selection and screened for Pr55gag expression. FIG. 14.2. Theviruses vgagl.l, and vgagl.2, e.g., vgag2.1 and 2.2, respectively werederived from the same initial plaques. All plaque isolates expressed astrong band in the 55 kDa region (p55). FIG. 14.2. The blot shows inaddition, a similar virus designated VVKI and described by Karacostas etal. (1989), which was kindly provided by B. Moss, NIH, Bethesda, Md.This virus has a P7.5 promoter-gag/pol precurser gene cassetteincorporated into the viral tk locus. Since infection was effected underthe same conditions in the same cell line, a direct comparison of theSep-gag and the P7.5-gag (VVKI) is possible. The Sep-gag constructsexpress at least fiveto ten-fold higher levels.

Structure of the chimeric viruses: To confirm the theoretical structuresof the chimeric viruses, Southern blot analyses were carried out. TotalDNA's from CV-1 cells infected with the respective viruses wereprepared, digested with HindIII and subjected to a Southern blotanalysis with a gag gene probe pgag(2) see FIG. 14.1) and the Not-regionprobe (see Example 13). With the gag gene probe, three fragments of12.8, 0.8 and 0.6 kb were expected for an insertion in the‘b’-orientation (direction of transcription of the Sep promoter is fromright to left). As shown in FIG. 14.3, all isolates examined had‘b’-orientation inserts. With the Not-region probe, two fragments of12.8 and 2.3 kb were expected and found. FIG. 14.4. As hybridizationcontrols, the HindIII (H) and Asp718 (A) fragments of plasmid psep-gagwere included in the Southern blot analysis. FIGS. 14.3 and 14.4.

Interestingly, only the ‘b’-orientation was detected (as in the case ofthe Sep-gpl6O constructs of Example 13), indicating that the strong Seppromoter must have the same orientation as the adjacent gene cluster.These findings highlight again the advantages of the direct cloningmethod. Insertion of this construct with a conventional insertionvector, directing the insert in a specific orientation, probably wouldhave failed if the wrong direction of orientation was chosen.

Further expression studies with the chimeric viruses vgag(l) andvgag(2): Based on the characterization steps described above, the virusvgag(l), derived from vgagl.2 after one more plaque purification step,and the virus vgag(2), derived from a second round of screening forhigh-expression isolates, were grown to high titers. With these twoviruses and the virus VVKI, a comparison of the gag-specific expressionlevels on CV-1 and Vero cells was performed. FIG. 14.5.

The viruses vgag(1) and vgag(2) show about the same levels of expressionin both cell lines. Their expression levels are much higher, however,than in the control construct VVKI. FIG. 14.5. Interestingly, the gagprecursor expressed by the virus vgag(l) is more intensely processed ascompared to the virus vgag(2).

Materials and Methods: Construction of the plasmids. The HIV-1 MN gagopen reading frame was prepared as a 1.8 kb SacI/HincII fragment fromthe plasmid pMN-ST2, provided by Dr. M. Reitz, NCI, Bethesda, Md., andinserted into the SacI/HincII digested vector pbluescript SK-(Stratagene). The resulting plasmid was designated pgagMN(S/H). The5′-end of pgagMN(S/H) was modified by digestion with ClaI and SacI andinsertion of the annealed oligonucleotides P-gag(1) (SEQ ID NO:94):5′-ACC ATG GGT GCG AGA GCG TCG GTA TTA AGC GGG GGA GAA TTA GAT-3′; andP-gag(2) (SEQ ID NO:95): 5′-CGA TCT AAT TCT CCC CCG CTT AAT ACC GAC GCTCTC GCA CCC ATG GTA GC T-3′. This plasmid was designated pgag2. A 1.7 kbNcoI/HincII fragment of pgag2 was inserted into the NcoI/StuI digestedplasmid pSep(l), thereby creating the vector psep-gag. For theconstruction of chimeric viruses, the 3.1 kb Not fragment encompassingthe Sep-promoter/gag-gene/P7.5-promoter-gpt gene cassette was used.Construction of the chimeric viruses was carried out as described above.

Western blot analysis gag-protein: Western blot analyses were performedessentially as described by Towbin et al. (1979). The first antibody wasa sheep anti-p24 antibody (Accurate Chemical & Scientific Corporation;Westbury, N.Y., #BOK-D7320) used at a 1:500 dilution. The secondantibody was a donkey anti-sheep IgG, coupled with alkaline phosphatase(obtained from Serotec, Inc.) used at a 1:1000 dilution. The reagents(BCIP and NBT) and staining protocols were from Promega, Inc.

EXAMPLE 15 Construction of the chimeric vaccinia virus vgagpol andexpression of recombinant HIV-1 gag-pol gene products includingpseudoparticles

Human immunodeficiency virus type I (HIV-1) contains an RNA genome thatencodes gag, pol, and env proteins, as well as additional regulatoryproteins. The primary gag translation product is a 55-kDa precursor, p55gag, that is processed into the major core proteins p24, pl7, and pl5 byproteolysis. The pol open reading frame encodes the protease, reversetranscriptase, and integrase. For a review, see Levy, J. A., Microbiol.Rev. 57:183-289 (1993).

Expression of the products of the pol gene requires a relativelyinefficient ribosomal frame shifting event within the gag gene thatleads to the formation of small amounts of the putative gag-polprecursor which is a protein of about 160 kDa. Jacks et al. (1988). Thepredominant intracellular polypeptides produced in CV-1 cells infectedwith a vaccinia virus carrying the gag-pol gene were p55, p41, p24 andp17. Reverse transcriptase activity was detected in cellularsupernatants and could be concentrated by centrifugation indicating thatpseudoparticles had formed. Karakostas et al. (1989).

Vaccines employing inactivated HIV-1 particles are considered a viableapproach in vaccine development. Since they are derived from infectiousHIV they pose many risks, e.g., during manufacture, incomplete virusinactivation and the existence of infectious residual HIV-1 genomic RNA.HIV-1 pseudoparticles are structurally very similar to normal HIVparticles and are efficient immunogens. There have been many reports onthe expression of HIV-1 pseudoparticles. See, for example, Gheysen etal. (1988); Hu et al. (1990); Karacostas et al. (1989). The problem ofthe low level of antigen expression, however, remains unsolved.

We have now expressed HIV-1 gag-pol genes under the control of thestrong early/late hybrid poxvirus promoter Sep and obtained very highexpression levels of the gag-pol gene products. A direct comparison withthe vaccinia recombinant vVKI of Karakostas et al. (1989) showed thatthe Sep-gag-pol constructs obtained by direct molecular cloning into theviral NotI site show an estimated ten-fold higher expression level and,therefore, are better candidates for the expression of HIV-1 gag-polgene products including pseudoparticles.

Construction of the plasmid pSep-gagpolIIIB and of the chimeric virusesvgagpol 7, vgagpol9 and vgagpol10: The HIV gag-pol sequences werederived from the plasmid pHB10 provided by R. Gallo, NCI Bethesda, Md.They were subcloned from pHB10 into pBluescript IISK and calledpgagpol 1. FIG. 15.1. To shorten the 5′-untranslated region of thegag-pol sequence and to subsequently introduce an NcoI site around thegag translation codon, the small ClaI-SacI fragment of pgagpol 1 wasreplaced by a fragment annealed from oligonucleotides P-gag(1) (SEQ IDNO:94) and P-gag(2) (SEQ ID NO:95) (see Example 14) resulting inpgagpol(2). The optimized gag-pol ORF was finally inserted into psep(l),resulting in pSep-gagpollIlB, from which the Sep-gagpol and P7.5-gptgenes can be excised as a NotI fragment.

Chimeric viruses were constructed as described (in Materials andMethods). Three isolates were further plaque-purified six times bygpt-selection and screened for gag-pol expression by Western blotanalysis. FIG. 15.2. The viruses vgagpol 7, vgagpol 9 and vgagpol 10express a strong band in the 55 kDa region and a weak one in the 160 kDaregion. FIG. 15.2. The blot shows, in addition, the expression level ofa similar virus, VVKI, described by Karakostas et al. (1989), which waskindly provided by B. Moss, NIH, Bethesda, Md. This virus hasincorporated a P7.5 promoter-gag-pol gene cassette into the viral tklocus. It expresses about ten-fold lower levels than the vgagpol virusesdescribed above.

Structure of the chimeric viruses: To confirm the structures of thechimeric viruses, Southern blot analyses were carried out. FIG. 15.3.Total DNA's from CV-1 cells infected with the respective viruses wereprepared, digested with HindIII, subjected to the Southern blotprocedure and hybridized to a gag-pol gene probe (see FIG. 15.1) and theNot-region probe (see Example 13). With the gag-pol gene probe, theexpected fragments of about 5.0, 0.9 and 0.6 kb were found, indicatingthat all three had the ‘a’-orientation (direction of transcription ofthe Sep promoter from left to right). The Asp718 and HindIII fragmentsof the plasmid pSep-gagpolIIIB were used as size markers. FIG. 15.3-ml,pSep-gagpolIIIB (HindIII); m2, pSep-gagpolIIIB (Asp7l8). The structureof the viruses also was confirmed with the Not-region probe (data notshown).

Further expression studies with the chimeric viruses vgag(1) 1.3 andvgag(2): Based on the screening and characterization steps describedabove, the virus vgagpol 7 was grown to high titer. Expression levels inCV-1 and Vero cells of the gag-pol gene products were confirmed. Inaddition, cellular supernatants were analysed. High levels of reversetranscriptase activity and of the gag-pol gene products were detectable.

Materials and Methods: Construction of the plasmids.

The intermediate plasmid pgag/pol(l) was constructed by insertion of a4.76 kb SacI/StuI fragment derived from the plasmid pBHIO in theSacI/HincII-cleaved vector pBluescript II SK- (Stratagene). Ratner etal. (1986); obtained from R. Gallo. The 5′-end of the gag gene wasmodified by removal of a 149 bp SacI/ClaI fragment and insertion of theannealed oligonucleotides P-gag(1) (SEQ ID NO: 94): 5′-ACCATGGGTGCGAGAGCGTC GGTATTAAGC GGGGGAGAAT TAGAT-3′; and P-gag(2) (SEQ ID NO:95):5′-CGATCTAATT CTCCCCCGCT TAATACCGAC GCTCTCGCAC CCATGGTAGC T-3′. Theresulting plasmid was designated pgag/pol(2). A 4.4 kb gag-pol geneNcoI/NdeI fragment of pgag/pol (2) was treated with Klenow Polymeraseand inserted into the SnaBI linearised vector pSep(l). The gag-pol genecassette of the resulting plasmid pSep-gag/polIIIB was used for theconstruction of the chimeric virus vgag/pol.

Construction of the chimeric viruses: The cloning and in vivo packagingprocedures were carried out as described in Examples 9 and 3,respectively.

Western Blot Analysis of gag and pol proteins: The Western Blot analyseswere done essentially as described by Towbin et al. (1979). For analysisof the gag-proteins, the first antibody was a sheep anti-p24 antibody(Accurate Chemical & Scientific Corporation, Westbury, N.Y., #BOK-D7320)used at a 1:500 dilution. The second antibody was a donkey anti-sheepIgG coupled with alkaline phosphatase (obtained from Serotec, Inc.) usedat a 1:1000 dilution. For analysis of the pol proteins, the firstantibody was a monoclonal, mouse anti-reverse transcriptase(HIV-1_(IIIB)) antibody (ABT, #9002, BIO-TRADE) used at a 1:1000dilution (protein content 1 ng/ul). The second antibody was a goatantimouse IgG (H+L) alkaline phosphatase conjugate (BIO-RAD #170-6520).The reagents (BCIP and NBT) and staining protocols were from Promega,Inc.

Formation of Pseudoparticles

The virus vgagpolIIIB#9 was used to produce HIV-1 pseudoparticles inCV-1 or Vero cells. The pseudoparticles present in the cellularsupernants of infected cells were isolated by centrifugation techniques.CV-1 (or Vero) cells were infected with 0.01 pfu per cell and incubatedfor 3-4 days until the cytopathic effect was complete. The cellularsupernants clarified at 1000 g for 5 min were subsequently purified bytwo sucrose-gradient centrifugations as described by Karacostas et al.,Proc. Natl. Acad. Sci. USA 86:8964 (1989).

For the Western blot analysis the pellets were resuspended inSDS-containing lysis buffer. A similar banding pattern as shown in FIG.15.2 could be observed in the Western blot analysis indicating that thesedimenting material contained the expected antigenic composition.

For the vaccination studies, the pelleted pseudoparticles wereresuspended in PBS and treated with formalin to inactivate residualvaccinia infectivity. The pseudoparticles generated both humoral andcell mediated immune response in mice and rabbits and may therefore beuseful immunogens in the prophylaxis and immunotherapy of AIDS.

EXAMPLE 16 Construction of the chimeric fowlpox virus f-aMN andexpression of recombinant HIV gp160MN in chicken cells

In Example 12, the construction of fowlpox virus (FPV) chimerasexpressing gpl6O controlled by a synthetic late promoter are discussed.Since it is desirable not only to use the FPV gpl6O MN constructs forproduction purposes, but also as live vaccines for priming the immuneresponse in humans, new constructs that express gpl6O early and late inthe viral life cycle were made. The hybrid promoter Sep (see Example 13)was used for these new constructs.

Construction of the chimeric viruses of the f-aMN series: To constructthe viruses of the f-aMN series, the double gene cassette consisting ofthe P7.5-gpt selection marker and the Sep-gpl6O gene was excised as aNotI fragment out of the plasmid pSep-ST2 and inserted directly into theunique NotI site of the fowlpox virus strain f-TK2a. Since directcloning usually results in the appearance of two orientations of theinsert, a schematic outline of the structures surrounding the insertionsites for the two possible orientations is shown in FIG. 16.1. Theconstruction of the plasmid pSep-ST2 has been described in Example 13and is shown in FIG. 13.1. This plasmid contains the HIV-1 gpl6OMNsequences controlled by the strong, semi-synthetic fowlpox viruspromoter Sep and a selection cassette consisting of the P7.5 promotergpt gene. Falkner and Moss (1988).

Twelve gpt positive viruses were plaque purified three times andscreened for expression of the env protein by Western blotting. FIG.16.2. For this purpose, confluent monolayers of CEF's were infected withone plaque forming unit of the respective fowlpox virus and harvestedafter four days. Total cellular proteins were separated on a 7.5%polyacrylamide gel, transferred to a nitrocellulose filter and furtherprocessed according to a standard Western blotting protocol. SeeMaterials and Methods. All of chimeric viruses expressed the HIV-1 gpl6Oproduct. FIG. 16.2. The viruses f-aMN4#3 and f-aMN6#20 were finallychosen on the basis of their high expression levels for large scalepurification and further characterization.

Fowlpox virus, although incapable of forming progeny virus in mammaliancells, does induce expression of foreign genes. Taylor et al. (1988). Toestimate expression levels in mammalian cells, Vero and CV-1 cells wereinfected with f-aMN, grown for 48 and 72 hours and analyzed by Westernblot analysis. FIG. 16.3. Taking into account that Western blotting is arelatively insensitive method, these experiments show that, in spite ofinfecting a non-avian host, relatively high levels of gpl6O wereobserved. Expression in CV-1 cells was slightly higher than in Verocells. Up to now, expression of foreign genes induced by FPV inmammalian cells could only be demonstrated by highly sensitive methodssuch as immunoprecipitations and immunofluorescence, underscoring theunexpected nature of the high efficiency seen with the newSep-Promoter-gpl6O constructs. See Taylor et al. (1988).

Structure of the chimeric viruses: To confirm structures of the chimericviruses, Southern blot analyses were carried out. The total DNA of CEF'sinfected with the respective viruses was cleaved with PstI, separated onan agarose gel, transferred to a nitrocellulose membrane and hybridizedto the gpl6O gene probe pMNenvl. With this probe, in case of the‘a’-orientation, a 28.2 kb fragment is expected which became visible inall isolates examined. See FIGS. 16.1 and 16.4. For unknown reasons thiscloning step resulted in a preferred orientation, the ‘a’-orientation.In the ‘b’-orientation a PstI fragment of 3.O kb was expected. FIG.16.1. The correct insertion of the gpl6O sequences into fowlpox viruswas demonstrated by this analysis.

The viruses f-aMN4#3 and f-aMN6#20 were chosen for large scalepurification and further characterization. They were shown to be free ofthe parental virus f-TK2a. Immunization studies with the virus f-aMN4#3in chickens (Example 17), mice and rabbits are carried out.

Construction of the chimeric viruses: Viruses were constructedessentially described in Example 3. Western blot analysis of gpl6O wereperformed as described in Example 13.

EXAMPLE 17 Immunization studies with f-aMN

The chimeric fowlpoxvirus f-aMN, which is derived from the highlyattenuated FPV vaccine strain HP1.441 of Mayr and Malicki (1966),expresses HIV-1 gpl6OMN. Seroconversion experiments with 12 week-oldSPF-chickens (Charles River, WIGA) were conducted to determine theimmunizing properties of this virus strain. From the literature, it isknown that FPV induces a weak humoral but a strong cell-mediatedimmunity. Mayr and Malicki (1966). This particular seroconversionexperiment was designed to examine the priming effects of the live virusf-aMN. Doses ranging from 10⁵ pfu to 10⁷ pfu per animal were used forimmunization.

The experimental design of the immunization study is outlined in Table17. Doses ranging from 10⁵ pfu to 10⁷ pfu per animal were used forimmunization. A second immunization was given after 3 weeks. A boosterimmunization of 50 ug per animal subunit gpl6OMN was given after another3 weeks. Antibody development against gpl6O was analyzed by Westernblotting and ELISA.

Preparation of the virus vaccine: The virus f-aMN was grown on chickenembryo fibroblasts and purified as described in U.S. Ser. No.07/882,768, hereby incorporated by reference.

‘Surf’ Western blot analysis: Total cellular proteins of HIV-1-infectedH9 cells were separated on a preparative polyacrylamide gel and blottedonto a nitrocellulose filter. Individual wells of a SURF-blot apparatus(Idea Scientific Co., Minneapolis, Minn.) were filled with differentdilutions of the respective serum sample and incubated for 1 hour.Further incubations were performed as described in Example 13.

TABLE 17 Vaccination schedule (12 week old chickens) 1. f-aMN: sucrosepurified viruses*, band, aliquots diluted in PBS to the respectivetiters (original titer: 1.2 × 10⁹) 2. HPI.441: (control)sucrose-purified viruses, band; aliquots diluted in PBS to therespective titer (original titer: 5 × 10⁹) day 0: a) bleed #1, 1 ml peranimal; pool blood b) injection of chickens (i.v. in wing vein) with 0.5ml of the respective virus dilution (group A) 5 chicks with 10⁵ pfu peranimal of f-aMN (group B) 5 chicks with 10⁶ pfu per animal of f-aMN(group C) 5 chicks with 10⁷ pfu per animal of f-aMN (group D) 5 chickswith 10⁷ pfu per animal of HP1.441 day 21: a) bleed #2 (1 ml per animal;pool blood) b) boost (same schedule as day 0) day 35: a) bleed #3 (i mlper animal; pool blood) b) boost with purified 50 ug gpl6OMN day 50: a)bleed #4 (total blood; end of experiment). *- vaccine stocks areprovided as frozen aliquots (−80° C.) of an appropriate size in PBS;vaccine stocks are vortexed prior to use.

The chicken sera of each group was pooled and examined in threedifferent ELISA assays: a gp160MN-strain specific ELISA, agp160IIIB-strain specific ELISA and a whole-virus ELISA. Table 18outlines the results of the immunization experiments.

TABLE 18 Elisa - Titer of pooled chicken sera after vaccination Day 2135 0 2. Infection Immunization 50 1. Infection with Fowlpox with End ofInfection with Fowlpox (Booster) 50 μg GP160 MN Experiment log 10 ElisaElisa Elisa Elisa Titer GP160 HIV-1 GP160 HIV-1 GP160 HIV-1 GP160 HIV-1Group Virus (pfu) MN IIIB IIIB MN IIIB IIIB MN IIIB IIIB MN IIIB IIIB Af-aMN 5.0 < < < < < < < < < 2560 1230  640 B f-aMN 6.0 < < < < < < < < <1280 1280 1280 C f-aMN 7.0 < < < < < < 160 < < 5120 5120 2560 D HP1.4417.0 < < < < < < < < < < < < (Control)

As expected from the literature, the capacity of a vaccine strain toinduce a humoral immune response of a fowlpox virus, in this case of arecombinant fowlpox virus, is low. Only the highest dose (10⁷ pfu peranimal, given twice) resulted in a weak titer of 1:160 in thegp160MN-specific ELISA. Boosting with 50 μg of purified gp160MN,however, resulted in a dose-dependent increase of the titers reachingmaximum titers of 1:5120. Even after priming with HP1.441 wild-typevirus, no seroconversion was obtained after a single dose of the gp160subunit vaccine (Table 18), confirming the capacity of the f-aMN virusto efficiently prime humoral immune reactions. Interestingly though,these antibodies cross-reacted with the HIV-1 IIIB strain.

In order to examine the capacity of the vaccine strains to prime humoralimmune response in a non-avian species, rabbits were selected for study.The vaccination scheme was similar to the chicken experiment and isshown in Table 19. The animals were vaccinated twice with the livevector and then boosted with the gp160 subunit. Higher doses of thevirus were used for immunization, because fowlpox does not replicate inrabbits. The results of the immunizations are shown in Table 20.Seroconversions (1:320) without boosting with gp160 subunit wereachieved only with the highest dose of 10⁸ pfu per animal. With the 10⁸dose, a strong ELISA titer (1:2560) developed after a single boost withgp160MN subunit in the MN-strain specific ELISA, and a somewhat lowertiter in the gp160IIIB ELISA. With the lower dose of 10⁷, seroconversioncould be demonstrated only after boosting with gp160 subunit reachingtiters of 1:160. With the control virus as priming agent, no antibodieswere demonstrated even after boosting with the gp160 subunit.

These results demonstrate the use of f-aMN as a priming vehicle inanimal systems such as chickens, in which fowlpox virus normallyreplicates, and in rabbits, in which FPV does not replicate. Rabbitshave long been used as models to evaluate human vaccines. Theexperiments with this non-avian species therefore strongly suggest thatother non-avian warm-blooded animals, e.g., the human, can be primedefficiently with the f-aMN virus.

TABLE 19 Vaccination schedule (10 week old rabbits) The vaccine stockswere provided as frozen aliquots (−80° C.) of an appropriate size inPBS; vaccine stocks were vortexed prior to use. 1. f-aMN Sucrosepurified (banded) virus, aliquots diluted in PBS to the respectivetiters (original titer: 1.2 × 10⁹ pfu/ml) 2. HP1.441 (control)sucrose-purified (banded) virus; aliquots diluted in PBS to therespective titer (original titer: 5 × 10⁹ pfu/ml) day 0: a) bleed #1, 1ml per animal; pool blood b) injection of rabbits (i.v. in ear vein)with 0.5 ml of the respective virus dilution 3 rabbits with 10⁶ pfu peranimal of f-aMN (Group Z) 3 rabbits with 10⁷ pfu per animal of f-aMN(Group Y) 3 rabbits with 10⁸ pfu per animal of f-aMN (Group X) 3 rabbitswith 10⁸ pfu per animal of HP1-441 (Group W) day 21: a) bleed #2 (1 mlper animal; pool blood) b) boost (same schedule as day 0) day 35: a)bleed #3 (1 ml per animal; pool blood) b) boost with purified 50 μggp160MN day 50: a) bleed #4 (total blood; end of experiment)

In order to reproduce and extend the animal priming studies, chickensand rabbits were immunized according to the vaccination schedules shownin Tables 21 and 23.

The immunizations of the chickens were carried out in a manner similarto that shown in Table 17, with the following modifications. Anadditional group of chickens (group E), the ‘PBS control’, was includedand a second immunization with gp160-subunit vaccine was given at day50. An additional set of titers were also determined after day 79. Inthese experiments the HIV-1 MN strain-specific ELISA was used to measurethe immune response.

The results of the chicken experiments confirm the high primingefficiency of the f-MN virus (Table 22). Two immunizations with thegp160 subunit vaccine alone (groups D and E, Table 22) resulted in a lowtiter of 1:100 after a long period of time. Two priming doses of thelive vaccine, followed by a booster injection of the gp160 subunitvaccine, even at the low titer of 10⁵, resulted in the high titer of1:10,000 (groups A-C, Table 22). A titer of this magnitude could not beobtained in any animal tested so far by conventional immunizationprocedures with the subunit gp160 vaccine.

The immunizations of the rabbits were carried out essentially as shownin Table 19, with the following modifications (See Table 23). Inaddition to the intravenous (i.v.) route, intramuscular (i.m.) andsubcutaneous (s.c.) injections were given. Additional groups of rabbits(groups V, Q, L), the ‘PBS controls’, were included and secondimmunizations with gp160-subunit vaccine were given at day 50. Finally,the dosage of the live vaccine was reduced (Table 23). The HIV-1MN-strain specific ELISA only was used to measure the immune response.

At the low dosage, for i.v. injections of the rabbits (first i.v.injection 10⁴ pfu and second i.v. injection 10⁶ pfu), no specificpriming effect was achieved by day 50 as compared to the controls (Table24, group Z). With the next dosage combination, first i.v. injection 10⁵pfu and second i.v. injection 10⁷ pfu (group Y, Table 24), the maximaltiter of 1:10,000 was achieved by day 50. The dosage (first i.v.injection 10⁶ pfu and second i.v. injection 10⁸ pfu; group X) did notimprove the titers significantly. Without priming with live virus, arise in ELISA titers was observed only after the second boosterinjection with the gpl60 subunit (Table 24, group W) at day 79.

The intramuscular injections also confirmed the priming potential of thef-aMN chimeric fowlpox virus. To achieve optimal titers, the dosagescheme first i.m. injection 10⁶ pfu and second i.m. injection 10⁸ pfu,was found to be optimal (Table 24, group S). The subcutaneous route alsoresulted in a measurable priming effect (Table 24).

gp160MN strain and IIIB strain specific ELISAS: Microtiter plates werecoated with purified gp160IIIB or gp160MN at a concentration of 5 μg/ml.After overnight incubation at 4° C., the plates were washed five timeswith PBS containing 0.05% Tween-20. Serum samples were serially dilutedin PBS containing 0.5% Tween and 1% serum proteins, beginning with a1:80 dilution. One hundred microliters of each sample were transferredto the coated plates. After an incubation of 1 hour at 37° C., theplates were washed five times with PBS-Tween solution and incubated foranother hour with 100 μl horseradish peroxidase-conjugated anti-IgG perwell. After washing five times with PBS-Tween, 100 μl ofO-phenylenediamine dihydrochlorate was added to each well. The colorreaction was stopped by the addition of 5M H₂SO₄ and the absorbance wasmeasured at 495 nm with a microplate spectrophotometer.

Whole Virus ELISA: The pooled sera were tested for whole HIV-1 viruswith the whole virus kit of Behring Enzygnost® as recommended by themanufacturer.

TABLE 20 Elisa - Titer of pooled rabbit sera after vaccination Day 21 350 2. Infection Immunization 50 1. Infection with Fowlpox with End ofInfection with Fowlpox (Booster) 50 μg GP160 MN Experiment log 10 ElisaElisa Elisa Elisa Titer GP160 HIV-1 GP160 HIV-1 GP160 HIV-1 GP160 HIV-1Group Virus (pfu) MN IIIB IIIB MN IIIB IIIB MN IIIB IIIB MN IIIB IIIB Xf-aMN #311 8.0 < < < < < < 320 320 < 2560 1280 160 Y f-aMN #311 7.0 < << < < < < < <  160  160 < Z f-aMN #311 6.0 < < < < < < < < < < < < WHP1.441 8.0 < < < < < < < < < < < < (Control)

TABLE 21 Vaccination Schedule of Chickens Each group consisted of sixchickens; the f-MN vaccine (first immunization and booster 1) were givenintravenous (iv) into the wing vein; boosters 2 and 3 consisted of 50 uggp160 MN in 0.5 ml solution and alum as an adjuvant; intramuscularinjections (im) were given at two sites into the left and right thighs;group first immunization and booster 1 group A iv per animal 0.5 ml 10⁵pfu f-aMN group B iv per animal 0.5 ml 10⁶ pfu f-aMN group C iv peranimal 0.5 ml 10⁷ pfu f-aMN group D iv per animal 0.5 ml 10⁷ HP1.441(wildtype) group E iv per animal 0.5 ml PBSA time schedule forvaccinations and blood samples day −7 pre-vaccination blood sample day 0first immunization day 21 Booster 1 and blood sample day 35 Booster 2and blood sample day 50 Booster 3 and blood sample day 79 blood sample

TABLE 22 Elisa Titers of Pooled Chicken Sera After Vaccination GP 160MN - Elisa-Titer Day 21 35 50 0 Booster 1 Booster 2 Booster 3 Infection(1. Infection (2. Infection (Immunisation (Immunisation 79 log 10 titer(pfu) with Fowlpox) with Fowlpox) with 50 μg with 50 μg End of GroupVirus Day 0 Day 21 (i.v.) (i.v.) GP 160 MN/MCC) GP 160 MN/MCC)Experiment A f-aMN 5.0 5.0 < < < 10000 10000 B f-aMN 6.0 5.0 < < < 1000010000 C f-aMN 7.0 7.0 < < 100 10000 10000 D HP1.441 7.0 7.0 < < < <  100(Control) E PBS — — < < < <  100 (Control) < = <1:100

TABLE 23 Vaccination Schedule of 10 Week Old Rabbits Boosters 2 and 3consisted of 50 μg gp160 MN in 0.5 ml solution and alum as an adjuvant;intravenous injections (iv) were given into the ear vein; intramuscularinjections (im) were given at two sites into the left and right thighs;subcutanceous injections (sc): same site as im injections Group FirstImmunization Booster 1 Group Z iv 0.5 ml 10⁴ pfu f-aMN 0.5 ml 10⁶ pfuf-aMN Group Y iv 0.5 ml 10⁵ pfu f-aMN 0.5 ml 10⁷ pfu f-aMN Group X iv0.5 ml 10⁶ pfu f-aMN 0.5 ml 10⁸ pfu f-aMN Group W iv 0.5 ml 10⁶ HP1.4410.5 ml 10⁶ HP1.441 Group V iv 0.5 ml PBS 0.5 ml PBS Group U im 0.5 ml10⁴ pfu f-aMN 0.5 ml 10⁶ pfu f-aMN Group T im 0.5 ml 10⁴ pfu f-aMN 0.5ml 10⁷ pfu f-aMN Group S im 0.5 ml 10⁶ pfu f-aMN 0.5 ml 10⁸ pfu f-aMNGroup R im 0.5 ml 10⁶ pfu f-aMN 0.5 ml 10⁸ pfu HP1.441 Group Q im 0.5 mlPBS 0.5 ml PBS Group P sc 0.5 ml 10⁴ pfu f-aMN 0.5 ml 10⁶ pfu f-aMNGroup O sc 0.5 ml 10⁵ pfu f-aMN 0.5 ml 10⁷ pfu f-aMN Group N sc 0.5 ml10⁶ pfu f-aMN 0.5 ml 10⁸ pfu f-aMN Group M sc 0.5 ml 10⁶ HP1.441 0.5 ml10⁸ HP1.441 Group L sc 0.5 ml PBS 0.5 ml PBS Time Schedule forvaccinations and Blood Samples Day 7 Pre vaccination blood sample Day 0First immunization Day 21 Booster 1 and blood sample Day 35 Booster 2and blood sample Day 50 Booster 3 and blood sample Day 79 Blood sample

TABLE 24 Elisa Titers of Pooled Rabbits Sera After Vaccination GP 160MN - Elisa-Titer Day 35 50 21 Booster 2 Booster 3 0 Booster 1(Immunisation (Immunisation Infection (1. Infection (2. Infection with50 μg with 50 μg 79 log 10 titer (pfu) with Fowlpox) with Fowlpox) GP160 MN/MCC) GP 160 MN/MCC) End of Group Virus Day 0 Day 21 Application(i.v.) (i.v.) (i.m.) (i.m.) Experiment Z f-aMN 4.0 6.0 i.v. < < < <10000  Y f-aMN 5.0 7.0 i.v. < < < 10000  10000  X f-aMN 6.0 8.0 i.v. < <100 10000  10000  W HP1.441 6.0 8.0 i.v. < < < < 1000 (Control) V PBS —— i.v. < < < < 1000 (Control) U f-aMN 4.0 6.0 i.m. < < < 100 1000 Tf-aMN 5.0 7.0 i.m. < < < 100 1000 S f-aMN 6.0 8.0 i.m. < < < 100 10000 R HP1.441 6.0 8.0 i.m. < < < 100 1000 (Control) Q PBS — — i.m. < < < <1000 (Control) P f-aMN 4.0 6.0 s.c. < < < 100 1000 O f-aMN 5.0 7.0 s.c.< < < 100 1000 N f-aMN 6.0 8.0 s.c. < < < 100 1000 M HP1.441 6.0 8.0s.c. < < < < 1000 (Control) L PBS — — s.c. < < < < 1000 (Control)

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 95(2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pN2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TCAAGCTTAT CGATACCGTCGCGGCCGCGA CCTCGAGGGG GGGCCCGG 48 (2) INFORMATION FOR SEQ ID NO:2: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1133 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: pN2-gpta (xi) SEQUENCE DESCRIPTION: SEQ IDNO:2: CTAGAACTAG TGGATCCCCC AACTTAAGGG TACCGCCTCG ACATCTATAT ACTATATAGT60 AATACCAATA CTCAAGACTA CGAAACTGAT ACAATCTCTT ATCATGTGGG TAATGTTCTC 120GATGTCGAAT AGCCATATGC CGGTAGTTGC GATATACATA AACTGATCAC TAATTCCAAA 180CCCACCCGCT TTTTATAGTA AGTTTTTCAC CCATAAATAA TAAATACAAT AATTAATTTC 240TCGTAAAAGT AGAAAATATA TTCTAATTTA TTGCACGGTA AGGAAGTAGA ATCATAAAGA 300ACAGTGACGG ATGATCCCCA AGCTTGGACA CAAGACAGGC TTGCGAGATA TGTTTGAGAA 360TACCACTTTA TCCCGCGTCA GGGAGAGGCA GTGCGTAAAA AGACGCGGAC TCATGTGAAA 420TACTGGTTTT TAGTGCGCCA GATCTCTATA ATCTCGCGCA ACCTATTTTC CCCTCGAACA 480CTTTTTAAGC CGTAGATAAA CAGGCTGGGA CACTTCACAT GAGCGAAAAA TACATCGTCA 540CCTGGGACAT GTTGCAGATC CATGCACGTA AACTCGCAAG CCGACTGATG CCTTCTGAAC 600AATGGAAAGG CATTATTGCC GTAAGCCGTG GCGGTCTGGT ACCGGGTGCG TTACTGGCGC 660GTGAACTGGG TATTCGTCAT GTCGATACCG TTTGTATTTC CAGCTACGAT CACGACAACC 720AGCGCGAGCT TAAAGTGCTG AAACGCGCAG AAGGCGATGG CGAAGGCTTC ATCGTTATTG 780ATGACCTGGT GGATACCGGT GGTACTGCGG TTGCGATTCG TGAAATGTAT CCAAAAGCGC 840ACTTTGTCAC CATCTTCGCA AAACCGGCTG GTCGTCCGCT GGTTGATGAC TATGTTGTTG 900ATATCCCGCA AGATACCTGG ATTGAACAGC CGTGGGATAT GGGCGTCGTA TTCGTCCCGC 960CAATCTCCGG TCGCTAATCT TTTCAACGCC TGGCACTGCC GGGCGTTGTT CTTTTTAACT 1020TCAGGCGGGT TACAATAGTT TCCAGTAAGT ATTCTGGAGG CTGCATCCAT GACACAGGCA 1080AACCTGAGCG AAACCCTGTT CAAACCCCGC TTTGGGCTGC AGGAATTCGA TAT 1133 (2)INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1133 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pN2-gptb (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CTAGAACTAGTGGATCCCCC AAAGCGGGGT TTGAACAGGG TTTCGCTCAG GTTTGCCTGT 60 GTCATGGATGCAGCCTCCAG AATACTTACT GGAAACTATT GTAACCCGCC TGAAGTTAAA 120 AAGAACAACGCCCGGCAGTG CCAGGCGTTG AAAAGATTAG CGACCGGAGA TTGGCGGGAC 180 GAATACGACGCCCATATCCC ACGGCTGTTC AATCCAGGTA TCTTGCGGGA TATCAACAAC 240 ATAGTCATCAACCAGCGGAC GACCAGCCGG TTTTGCGAAG ATGGTGACAA AGTGCGCTTT 300 TGGATACATTTCACGAATCG CAACCGCAGT ACCACCGGTA TCCACCAGGT CATCAATAAC 360 GATGAAGCCTTCGCCATCGC CTTCTGCGCG TTTCAGCACT TTAAGCTCGC GCTGGTTGTC 420 GTGATCGTAGCTGGAAATAC AAACGGTATC GACATGACGA ATACCCAGTT CACGCGCCAG 480 TAACGCACCCGGTACCAGAC CGCCACGGCT TACGGCAATA ATGCCTTTCC ATTGTTCAGA 540 AGGCATCAGTCGGCTTGCGA GTTTACGTGC ATGGATCTGC AACATGTCCC AGGTGACGAT 600 GTATTTTTCGCTCATGTGAA GTGTCCCAGC CTGTTTATCT ACGGCTTAAA AAGTGTTCGA 660 GGGGAAAATAGGTTGCGCGA GATTATAGAG ATCTGGCGCA CTAAAAACCA GTATTTCACA 720 TGAGTCCGCGTCTTTTTACG CACTGCCTCT CCCTGACGCG GGATAAAGTG GTATTCTCAA 780 ACATATCTCGCAAGCCTGTC TTGTGTCCAA GCTTGGGGAT CATCCGTCAC TGTTCTTTAT 840 GATTCTACTTCCTTACCGTG CAATAAATTA GAATATATTT TCTACTTTTA CGAGAAATTA 900 ATTATTGTATTTATTATTTA TGGGTGAAAA ACTTACTATA AAAAGCGGGT GGGTTTGGAA 960 TTAGTGATCAGTTTATGTAT ATCGCAACTA CCGGCATATG GCTATTCGAC ATCGAGAACA 1020 TTACCCACATGATAAGAGAT TGTATCAGTT TCGTAGTCTT GAGTATTGGT ATTACTATAT 1080 AGTATATAGATGTCGAGGCG GTACCCTTAA GTTGGGCTGC AGGAATTCGA TAT 1133 (2) INFORMATION FORSEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: pHindJ-2 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:4: CGCATTTTCT AACGTGATGG GATCCGTTAACTCGCGAGAA TTCTGTAGAA AGTGTTACAT 60 CGACTC 66 (2) INFORMATION FOR SEQ IDNO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 127 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: pHindJ-3 (xi) SEQUENCE DESCRIPTION:SEQ ID NO:5: CGCATTTTCT AACGTGATGG GATCCGGCCG GCTAGGCCGC GGCCGCCCGGGTTTTTATCT 60 CGAGACAAAA AGACGGACCG GGCCCGGCCA TATAGGCCCA ATTCTGTAGAAAGTGTTACA 120 TCGACTC 127 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 115 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: pA0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:AGGGAACAAA AGCTGGAGCT AGGCCGGCTA GGCCGCGGCC GCCCGGGTTT TTATCTCGAG 60ACAAAAAGAC GGACCGGGCC CGGCCATATA GGCCAGTACC CAATTCGCCC TATAG 115 (2)INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:103 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pA1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CGGCCGCCCG GGTTTTTATCTCGACATATG CTGCAGTTAA CGAATTCCAT GGGGATCCGA 60 TATCAAGCTT AGGCCTGTCGACGTCGAGAC AAAAAGACGG ACC 103 (2) INFORMATION FOR SEQ ID NO:8: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 103 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: pA2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:CGGCCGCCCG GGTTTTTATC TCGACGTCGA CAGGCCTAAG CTTGATATCG GATCCCCATG 60GAATTCGTTA ACTGCAGCAT ATGTCGAGAC AAAAAGACGG ACC 103 (2) INFORMATION FORSEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 213 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: pA1-S1 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:9: CCCGGGTTTT TATCTCGACA TACGGCTTGG TATAGCGGACAACTAAGTAA TTGTAAAGAA 60 GAAAACGAAA CTATCAAAAC CGTTTATGAA ATGATAGAAAAAAGAATATA AATAATCCTG 120 TATTTTAGTT TAAGTAACAG TAAAATAATG AGTAGAAAATACTATTTTTT ATAGCCTATA 180 AATCATGAAT TCGGATCCGA TATCAAGCTT AGG 213 (2)INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:215 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pA2-S1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CAGGCCTAAGCTTGATATCG GATCCGAATT CATGATTTAT AGGCTATAAA AAATAGTATT 60 TTCTACTCATTATTTTACTG TTACTTAAAC TAAAATACAG GATTATTTAT ATTCTTTTTT 120 CTATCATTTCATAAACGGTT TTGATAGTTT CGTTTTCTTC TTTACAATTA CTTAGTTGTC 180 CGCTATACCAAGCCGTATGT CGAGACAAAA AGACG 215 (2) INFORMATION FOR SEQ ID NO:11: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 88 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: pA1-S2 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:11: TCTCGACATA TGCTGCAGTT GGGAAGCTTT TTTTTTTTTT TTTTTTTGGC ATATAAATAG60 GCTGCAGGAA TTCCATGGGG ATCCGATA 88 (2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 92 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: pA2-S2 (xi) SEQUENCE DESCRIPTION: SEQID NO:12: TTGATATCGG ATCCCCATGG AATTCCTGCA GCCTATTTAT ATGCCAAAAAAAAAAAAAAA 60 AAAAAGCTTC CCAACTGCAG CATATGTCGA GA 92 (2) INFORMATION FORSEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 127 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: pN2gpt-S3A (fig. 4.7)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TACCCTTAAG TTGGGCTGCAGAAGCTTTTT TTTTTTTTTT TTTTTGGCAT ATAAATGAAT 60 TCCATGGCCC GGGAAGGCCTCGGACCGGGC CCGGCCATAT AGGCCAGCGA TACCGTCGCG 120 GCCGCGA 127 (2)INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:134 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pN2gpt-S4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TACCCTTAAGTTGGGCTGCA GAAGCTTTTT TTTTTTTTTT TTTTTGGCAT ATAAATCGTT 60 AACGAATTCCATGGCCCGGG AAGGCCTCGG ACCGGGCCCG GCCATATAGG CCAGCGATAC 120 CGTCGCGGCCGCGA 134 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1988 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pA1S1-PT (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: TTTTATAGCCTATAAATCAT GAATTCCGCG CACGTCCGAG GCTTGCAGCT GCCTGGCTGC 60 CTGGCCCTGGCTGCCCTGTG TAGCCTTGTG CACAGCCAGC ATGTGTTCCT GGCTCCTCAG 120 CAAGCACGGTCGCTGCTCCA GCGGGTCCGG CGAGCCAACA CCTTCTTGGA GGAGGTGCGC 180 AAGGGCAACCTAGAGCGAGA GTGCGTGGAG GAGACGTGCA GCTACGAGGA GGCCTTCGAG 240 GCTCTGGAGTCCTCCACGGC TACGGATGTG TTCTGGGCCA AGTACACAGC TTGTGAGACA 300 GCGAGGACGCCTCGAGATAA GCTTGCTGCA TGTCTGGAAG GTAACTGTGC TGAGGGTCTG 360 GGTACGAACTACCGAGGGCA TGTGAACATC ACCCGGTCAG GCATTGAGTG CCAGCTATGG 420 AGGAGTCGCTACCCACATAA GCCTGAAATC AACTCCACTA CCCATCCTGG GGCCGACCTA 480 CAGGAGAATTTCTGCCGCAA CCCCGACAGC AGCAACACGG GACCATGGTG CTACACTACA 540 GACCCCACCGTGAGGAGGCA GGAATGCAGC ATCCCTGTCT GTGGCCAGGA TCAAGTCACT 600 GTAGCGATGACTCCACGCTC CGAAGGCTCC AGTGTGAATC TGTCACCTCC ATTGGAGCAG 660 TGTGTCCCTGATCGGGGGCA GCAGTACCAG GGGCGCCTGG CGGTGACCAC ACATGGGCTC 720 CCCTGCCTGGCCTGGGCCAG CGCACAGGCC AAGGCCCTGA GCAAGCACCA GGACTTCAAC 780 TCAGCTGTGCAGCTGGTGGA GAACTTCTGC CGCAACCCAG ACGGGGATGA GGAGGGCGTG 840 TGGTGCTATGTGGCCGGGAA GCCTGGCGAC TTTGGGTACT GCGACCTCAA CTATTGTGAG 900 GAGGCCGTGGAGGAGGAGAC AGGAGATGGG CTGGATGAGG ACTCAGACAG GGCCATCGAA 960 GGGCGTACCGCCACAAGTGA GTACCAGACT TTCTTCAATC CGAGGACCTT TGGCTCGGGA 1020 GAGGCAGACTGTGGGCTGCG ACCTCTGTTC GAGAAGAAGT CGCTGGAGGA CAAAACCGAA 1080 AGAGAGCTCCTGGAATCCTA CATCGACGGG CGCATTGTGG AGGGCTCGGA TGCAGAGATC 1140 GGCATGTCACCTTGGCAGGT GATGCTTTTC CGGAAGAGTC CCCAGGAGCT GCTGTGTGGG 1200 GCCAGCCTCATCAGTGACCG CTGGGTCCTC ACCGCCGCCC ACTGCCTCCT GTACCCGCCC 1260 TGGGACAAGAACTTCACCGA GAATGACCTT CTGGTGCGCA TTGGCAAGCA CTCCCGCACC 1320 AGGTACGAGCGAAACATTGA AAAGATATCC ATGTTGGAAA AGATCTACAT CCACCCCAGG 1380 TACAACTGGCGGGAGAACCT GGACCGGGAC ATTGCCCTGA TGAAGCTGAA GAAGCCTGTT 1440 GCCTTCAGTGACTACATTCA CCCTGTGTGT CTGCCCGACA GGGAGACGGC AGCCAGCTTG 1500 CTCCAGGCTGGATACAAGGG GCGGGTGACA GGCTGGGGCA ACCTGAAGGA GACGTGGACA 1560 GCCAACGTTGGTAAGGGGCA GCCCAGTGTC CTGCAGGTGG TGAACCTGCC CATTGTGGAG 1620 CGGCCGGTCTGCAAGGACTC CACCCGGATC CGCATCACTG ACAACATGTT CTGTGCTGGT 1680 TACAAGCCTGATGAAGGGAA ACGAGGGGAT GCCTGTGAAG GTGACAGTGG GGGACCCTTT 1740 GTCATGAAGAGCCCCTTTAA CAACCGCTGG TATCAAATGG GCATCGTCTC ATGGGGTGAA 1800 GGCTGTGACCGGGATGGGAA ATATGGCTTC TACACACATG TGTTCCGCCT GAAGAAGTGG 1860 ATACAGAAGGTCATTGATCA GTTTGGAGAG TAGGGGGCCA CTCATATTCT GGGCTCCTGG 1920 AACCAATCCCGTGAAAGAAT TATTTTTGTG TTTCTAAAAC TAGAATTCGG ATTCGATATC 1980 AAGCTTAG1988 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: odN1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GGCCAGGCCTTTTAAATTAA GATATC 26 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 111 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: pN2gpt-GPg (xi) SEQUENCE DESCRIPTION: SEQID NO:17: TTTTTGGCAT ATAAATCGTT CCAGTCCCAA AATGTAATTG GACGGGAGACAGAGTGACGC 60 ACGCGGCCGC TCTAGAACTA GTGGATCCCC CAACGAATTC CATGGCCCGG G111 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 2296 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pN2gpt-LPg (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATAAATCGTTAACGAATTCC ATGGAACATA AGGAAGTGGT TCTTCTACTT CTTTTATTTC 60 TGAAATCAGGTCAAGGAAAA GTGTATCTCT CAGAGTGCAA GACTGGGAAT GGAAAGAACT 120 ACAGAGGGACGATGTCCAAA ACAAAAAATG GCATCACCTG TCAAAAATGG AGTTCCACTT 180 CTCCCCACAGACCTAGATTC TCACCTGCTA CACACCCCTC AGAGGGACTG GAGGAGAACT 240 ACTGCAGGAATCCAGACAAC GATCCGCAGG GGCCCTGGTG CTATACTACT GATCCAGAAA 300 AGAGATATGACTACTGCGAC ATTCTTGAGT GTGAAGAGGA ATGTATGCAT TGCAGTGGAG 360 AAAACTATGACGGCAAAATT TCCAAGACCA TGTCTGGACT GGAATGCCAG GCCTGGGACT 420 CTCAGAGCCCACACGCTCAT GGATACATTC CTTCCAAATT TCCAAACAAG AACCTGAAGA 480 AGAATTACTGTCGTAACCCC GATAGGGAGC TGCGGCCTTG GTGTTTCACC ACCGACCCCA 540 ACAAGCGCTGGGAACTTTGC GACATCCCCC GCTGCACAAC ACCTCCACCA TCTTCTGGTC 600 CCACCTACCAGTGTCTGAAG GGAACAGGTG AAAACTATCG CGGGAATGTG GCTGTTACCG 660 TTTCCGGGCACACCTGTCAG CACTGGAGTG CACAGACCCC TCACACACAT AACAGGACAC 720 CAGAAAACTTCCCCTGCAAA AATTTGGATG AAAACTACTG CCGCAATCCT GACGGAAAAA 780 GGGCCCCATGGTGCCATACA ACCAACAGCC AAGTGCGGTG GGAGTACTGT AAGATACCGT 840 CCTGTGACTCCTCCCCAGTA TCCACGGAAC AATTGGCTCC CACAGCACCA CCTGAGCTAA 900 CCCCTGTGGTCCAGGACTGC TACCACGGTG ATGGACAGAG CTACCGAGGC ACATCCTCCA 960 CCACCACCACAGGAAAGAAG TGTCAGTCTT GGTCATCTAT GACACCACAC CGGCACCAGA 1020 AGACCCCAGAAAACTACCCA AATGCTGGCC TGACAATGAA CTACTGCAGG AATCCAGATG 1080 CCGATAAAGGCCCCTGGTGT TTTACCACAG ACCCCAGCGT CAGGTGGGAG TACTGCAACC 1140 TGAAAAAATGCTCAGGAACA GAAGCGAGTG TTGTAGCACC TCCGCCTGTT GTCCTGCTTC 1200 CAGATGTAGAGACTCCTTCC GAAGAAGACT GTATGTTTGG GAATGGGAAA GGATACCGAG 1260 GCAAGAGGGCGACCACTGTT ACTGGGACGC CATGCCAGGA CTGGGCTGCC CAGGAGCCCC 1320 ATAGACACAGCATTTTCACT CCAGAGACAA ATCCACGGGC GGGTCTGGAA AAAAATTACT 1380 GCCGTAACCCTGATGGTGAT GTAGGTGGTC CCTGGTGCTA CACGACAAAT CCAAGAAAAC 1440 TTTACGACTACTGTGATGTC CCTCAGTGTG CGGCCCCTTC ATTTGATTGT GGGAAGCCTC 1500 AAGTGGAGCCGAAGAAATGT CCTGGAAGGG TTGTGGGGGG GTGTGTGGCC CACCCACATT 1560 CCTGGCCCTGGCAAGTCAGT CTTAGAACAA GGTTTGGAAT GCACTTCTGT GGAGGCACCT 1620 TGATATCCCCAGAGTGGGTG TTGACTGCTG CCCACTGCTT GGAGAAGTCC CCAAGGCCTT 1680 CATCCTACAAGGTCATCCTG GGTGCACACC AAGAAGTGAA TCTCGAACCG CATGTTCAGG 1740 AAATAGAAGTGTCTAGGCTG TTCTTGGAGC CCACACGAAA AGATATTGCC TTGCTAAAGC 1800 TAAGCAGTCCTGCCGTCATC ACTGACAAAG TAATCCCAGC TTGTCTGCCA TCCCCAAATT 1860 ATGTGGTCGCTGACCGGACC GAATGTTTCA TCACTGGCTG GGGAGAAACC CAAGGTACTT 1920 TTGGAGCTGGCCTTCTCAAG GAAGCCCAGC TCCCTGTGAT TGAGAATAAA GTGTGCAATC 1980 GCTATGAGTTTCTGAATGGA AGAGTCCAAT CCACCGAACT CTGTGCTGGG CATTTGGCCG 2040 GAGGCACTGACAGTTGCCAG GGTGACAGTG GAGGTCCTCT GGTTTGCTTC GAGAAGGACA 2100 AATACATTTTACAAGGAGTC ACTTCTTGGG GTCTTGGCTG TGCACGCCCC AATAAGCCTG 2160 GTGTCTATGTTCGTGTTTCA AGGTTTGTTA CTTGGATTGA GGGAGTGATG AGAAATAATT 2220 AATTGGACGGGAGACAGAGT GACGCACGCG GCCGCTCTAG AACTAGTGGA TCCCCCGGGA 2280 AGGCCTCGGACCGGGC 2296 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 56 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: pN2gpt-gp160 (xi) SEQUENCE DESCRIPTION: SEQID NO:19: TTTTTGGCAT ATAAATCGTT ATCCACCATG TAAGATAACG AATTCCATGG CCCGGG56 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 331 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pvWF (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: TTTTTTTTGGCATATAAATC GCGGCCGCGG GTGGTTGGTG GATGTCACAG CTTGGGCTTT 60 ATCTCCCCCAGCAGTGGGAT TCCACAGCCC CTGGGCTACA TAACAGCAAG ACAGTCCGGA 120 GCTGTAGCAGACCTGATTGA GCCTTTGCAG CAGCTGAGAG CATGGCCTAG GGTGGGCGGC 180 ACCATTGTCCAGCAGCTGAG TTTCCCAGGG ACCTTGGAGA TAGCCGCAGC CCTCATTTGC 240 AGGGGAAGATGTGAGGCTGC TGCAGCTGCA TGGGTGCCTG CTGCTGCCTG CCTTGGCCTG 300 ATGGCGGCCGCCCGGGTTTT TATCTCGAGA C 331 (2) INFORMATION FOR SEQ ID NO:21: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: pEcoK-dhr (xi) SEQUENCE DESCRIPTION: SEQ IDNO:21: ATTAGCGTCT CGTTTCAGAC GCGGCCGCGG TAATTAGATT CTCCCACATT 50 (2)INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1209 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pdhr-gpt (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ATTAGCGTCTCGTTTCAGAC GCGGCCGCTC TAGAACTAGT GGATCCCCCA ACTTAAGGGT 60 ACCGCCTCGACATCTATATA CTATATAGTA ATACCAATAC TCAAGACTAC GAAACTGATA 120 CAATCTCTTATCATGTGGGT AATGTTCTCG ATGTCGAATA GCCATATGCC GGTAGTTGCG 180 ATATACATAAACTGATCACT AATTCCAAAC CCACCCGCTT TTTATAGTAA GTTTTTCACC 240 CATAAATAATAAATACAATA ATTAATTTCT CGTAAAAGTA GAAAATATAT TCTAATTTAT 300 TGCACGGTAAGGAAGTAGAA TCATAAAGAA CAGTGACGGA TGATCCCCAA GCTTGGACAC 360 AAGACAGGCTTGCGAGATAT GTTTGAGAAT ACCACTTTAT CCCGCGTCAG GGAGAGGCAG 420 TGCGTAAAAAGACGCGGACT CATGTGAAAT ACTGGTTTTT AGTGCGCCAG ATCTCTATAA 480 TCTCGCGCAACCTATTTTCC CCTCGAACAC TTTTTAAGCC GTAGATAAAC AGGCTGGGAC 540 ACTTCACATGAGCGAAAAAT ACATCGTCAC CTGGGACATG TTGCAGATCC ATGCACGTAA 600 ACTCGCAAGCCGACTGATGC CTTCTGAACA ATGGAAAGGC ATTATTGCCG TAAGCCGTGG 660 CGGTCTGGTACCGGGTGCGT TACTGGCGCG TGAACTGGGT ATTCGTCATG TCGATACCGT 720 TTGTATTTCCAGCTACGATC ACGACAACCA GCGCGAGCTT AAAGTGCTGA AACGCGCAGA 780 AGGCGATGGCGAAGGCTTCA TCGTTATTGA TGACCTGGTG GATACCGGTG GTACTGCGGT 840 TGCGATTCGTGAAATGTATC CAAAAGCGCA CTTTGTCACC ATCTTCGCAA AACCGGCTGG 900 TCGTCCGCTGGTTGATGACT ATGTTGTTGA TATCCCGCAA GATACCTGGA TTGAACAGCC 960 GTGGGATATGGGCGTCGTAT TCGTCCCGCC AATCTCCGGT CGCTAATCTT TTCAACGCCT 1020 GGCACTGCCGGGCGTTGTTC TTTTTAACTT CAGGCGGGTT ACAATAGTTT CCAGTAAGTA 1080 TTCTGGAGGCTGCATCCATG ACACAGGCAA ACCTGAGCGA AACCCTGTTC AAACCCCGCT 1140 TTGGGCTGCAGGAATTCGAT ATCAAGCTTA TCGATACCGT CGCGGCCGCG GTAATTAGAT 1200 TCTCCCACA1209 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: odN2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GGCCGATATCTTAATTTAAA AGGCCT 26 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: odN3 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:24: CCAATGTTAC GTGGGTTACA TCAG 24 (2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: I-SceI linker 1 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:25: TAGGGATAAC AGGGTAAT 18 (2) INFORMATION FORSEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: I-SceI linker 2 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:26: ATTACCCTGT TATCCCTA 18 (2)INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: odS2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GTATAAAGTCCGACTATTGT TCT 23 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: odS3 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:28: TCTGAGGCCT AATAGACCTC TGTACA 26 (2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: SfiI(1) (xi) SEQUENCE DESCRIPTION:SEQ ID NO:29: GGCCGGCTAG GCC 13 (2) INFORMATION FOR SEQ ID NO:30: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: SfiI(2) (xi) SEQUENCE DESCRIPTION: SEQ IDNO:30: GGCCATATAG GCC 13 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: odTK1 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:31: GAGTCGATGT AACACTTTCT ACAGGATCCG TTAACTCGCG AGAATTCCAT CACGTTAGAA60 AATGCG 66 (2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: P-J(1) (xi) SEQUENCE DESCRIPTION: SEQ IDNO:32: GATCCGGCCG GCTAGGCCGC GGCCGCCCGG GTTTTTATCT CGAGACAAAA AGACGGACCG60 GGCCCGGCCA TATAGGCCC 79 (2) INFORMATION FOR SEQ ID NO:33: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 79 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: P-J(2) (xi) SEQUENCE DESCRIPTION: SEQ IDNO:33: AATTGGGCCT ATATGGCCGG GCCCGGTCCG TCTTTTTGTC TCGAGATAAA AACCCGGGCG60 GCCGCGGCCT AGCCGGCCG 79 (2) INFORMATION FOR SEQ ID NO:34: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: odTK2 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:34: AGAAGCCGTG GGTCATTG 18 (2) INFORMATION FOR SEQ ID NO:35: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: odTK3 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:35: TACCGTGTCG CTGTAACTTA C 21 (2) INFORMATION FOR SEQ ID NO:36: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 75 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: P-A(0.1) (xi) SEQUENCE DESCRIPTION: SEQ IDNO:36: AGGCCGGCTA GGCCGCGGCC GCCCGGGTTT TTATCTCGAG ACAAAAAGAC GGACCGGGCC60 CGGCCATATA GGCCA 75 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 83 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: P-A(0.2) (xi) SEQUENCE DESCRIPTION: SEQ IDNO:37: GTACTGGCCT ATATGGCCGG GCCCGGTCCG TCTTTTTGTC TCGAGATAAA AACCCGGGCG60 GCCGCGGCCT AGCCGGCCTA GCT 83 (2) INFORMATION FOR SEQ ID NO:38: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 55 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: P-artP(11) (xi) SEQUENCE DESCRIPTION: SEQID NO:38: GGCCACGTTT TTATGGGAAG CTTTTTTTTT TTTTTTTTTT TGGCATATAA ATCGC55 (2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: P-artP(12) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GGCCGCGATTTATATGCCAA AAAAAAAAAA AAAAAAAAGC TTCCCATAAA AACGT 55 (2) INFORMATION FORSEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 93 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: P-artP(8) (xi)SEQUENCE DESCRIPTION: SEQ ID NO:40: CGCTGGCCTA TATGGCCGGG CCCGGTCCGAGGCCTTCCCG GGCCATGGAA TTCATTTATA 60 TGCCAAAAAA AAAAAAAAAA AAAAGCTTCT GCA93 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: P-artP(10) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: CGCTGGCCTATATGGCCGGG CGTCCGAGGC CTTCCCGGGC CATGGAATTC GTTAACGATT 60 TATATGCCAAAAAAAAAAAA AAAAAAAAGC TTCTGCA 97 (2) INFORMATION FOR SEQ ID NO:42: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: oligonucleotide P-hr(3) (xi) SEQUENCEDESCRIPTION: SEQ ID NO:42: ATTAGCGTCT CGTTTCAGAC GCGGCCGCGG TAATTAGATTCTCCCACATT 50 (2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (xi)SEQUENCE DESCRIPTION: SEQ ID NO:43: CTAGCCCGGG 10 (2) INFORMATION FORSEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: P-P2 5′(1) (xi)SEQUENCE DESCRIPTION: SEQ ID NO:44: GTACGTACGG CTGCAGTTGT TAGAGCTTGGTATAGCGGAC AACTAAG 47 (2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: P-P2 3′(1) (xi) SEQUENCE DESCRIPTION: SEQID NO:45: TCTGACTGAC GTTAACGATT TATAGGCTAT AAAAAATAGT ATTTTCTACT 50 (2)INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: P-SM(2) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GTCTTGAGTATTGGTATTAC 20 (2) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: P-SM(3) (xi) SEQUENCE DESCRIPTION: SEQ IDNO:47: CGAAACTATC AAAACGCTTT ATG 23 (2) INFORMATION FOR SEQ ID NO:48:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: P-MN(1) (xi) SEQUENCE DESCRIPTION:SEQ ID NO:48: AGCTAGCTGA ATTCAGGCCT CATGAGAGTG AAGGGGATCA GGAGGAATTA TCA53 (2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: P-MN(2) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: CATCTGATGCACAAAATAGA GTGGTGGTTG 30 (2) INFORMATION FOR SEQ ID NO:50: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: P-Seq(2) (xi) SEQUENCE DESCRIPTION: SEQ IDNO:50: CTGTGGGTAC ACAGGCTTGT GTGGCCC 27 (2) INFORMATION FOR SEQ IDNO:51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: P-Seq(3) (xi) SEQUENCE DESCRIPTION:SEQ ID NO:51: CAATTTTTCT GTAGCACTAC AGATC 25 (2) INFORMATION FOR SEQ IDNO:52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: o-542 (xi) SEQUENCE DESCRIPTION: SEQID NO:52: CGATTACGTA GTTAACGCGG CCGCGGCCTA GCCGGCCATA AAAAT 45 (2)INFORMATION FOR SEQ ID NO:53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: o-544 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: CTAGATTTTTATGGCCGGCT AGGCCGCGGC CGCGTTAACT ACGTAAT 47 (2) INFORMATION FOR SEQ IDNO:54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: o-541 (xi) SEQUENCE DESCRIPTION: SEQID NO:54: CTTTTTCTGC GGCCGCGGAT ATGGCCCGGT CCGGTTAACT ACGTAGACGT 50 (2)INFORMATION FOR SEQ ID NO:55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:53 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: o-543 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: CTACGTAGTTAACCGGACCG GGCCATATAG GCCGCGGCCG CAGAAAAAGC ATG 53 (2) INFORMATION FORSEQ ID NO:56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 51 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: o-selPI (xi) SEQUENCEDESCRIPTION: SEQ ID NO:56: CGATAAAAAT TGAAATTTTA TTTTTTTTTT TTGGAATATAAATAAGGCCT C 51 (2) INFORMATION FOR SEQ ID NO:57: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: o-selPII (xi) SEQUENCE DESCRIPTION: SEQ IDNO:57: CATGGAGGCC TTATTTATAT TCCAAAAAAA AAAAATAAAA TTTCAATTTT TAT 53 (2)INFORMATION FOR SEQ ID NO:58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: o-830 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: TCGACTTTTT ATCA 14(2) INFORMATION FOR SEQ ID NO:59: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: o-857 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: TATGATAAAA AC 12(2) INFORMATION FOR SEQ ID NO:60: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: o-NcoI (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: GAGCAGAAGACAGTGGCCAT GGCCGTGAAG GGGATCAGGA 40 (2) INFORMATION FOR SEQ ID NO:61:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: o-NsiI (xi) SEQUENCE DESCRIPTION: SEQID NO:61: CATAAACTGA TTATATCCTC ATGCATCTGT 30 (2) INFORMATION FOR SEQ IDNO:62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4145 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: pS2gpt-S4 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:62: GTGGCACTTT TCGGGGAAAT GTGCGCGGAACCCCTATTTG TTTATTTTTC TAAATACATT 60 CAAATATGTA TCCGCTCATG AGACAATAACCCTGATAAAT GCTTCAATAA TATTGAAAAA 120 GGAAGAGTAT GAGTATTCAA CATTTCCGTGTCGCCCTTAT TCCCTTTTTT GCGGCATTTT 180 GCCTTCCTGT TTTTGCTCAC CCAGAAACGCTGGTGAAAGT AAAAGATGCT GAAGATCAGT 240 TGGGTGCACG AGTGGGTTAC ATCGAACTGGATCTCAACAG CGGTAAGATC CTTGAGAGTT 300 TTCGCCCCGA AGAACGTTTT CCAATGATGAGCACTTTTAA AGTTCTGCTA TGTGGCGCGG 360 TATTATCCCG TATTGACGCC GGGCAAGAGCAACTCGGTCG CCGCATACAC TATTCTCAGA 420 ATGACTTGGT TGAGTACTCA CCAGTCACAGAAAAGCATCT TACGGATGGC ATGACAGTAA 480 GAGAATTATG CAGTGCTGCC ATAACCATGAGTGATAACAC TGCGGCCAAC TTACTTCTGA 540 CAACGATCGG AGGACCGAAG GAGCTAACCGCTTTTTTGCA CAACATGGGG GATCATGTAA 600 CTCGCCTTGA TCGTTGGGAA CCGGAGCTGAATGAAGCCAT ACCAAACGAC GAGCGTGACA 660 CCACGATGCC TGTAGCAATG GCAACAACGTTGCGCAAACT ATTAACTGGC GAACTACTTA 720 CTCTAGCTTC CCGGCAACAA TTAATAGACTGGATGGAGGC GGATAAAGTT GCAGGACCAC 780 TTCTGCGCTC GGCCCTTCCG GCTGGCTGGTTTATTGCTGA TAAATCTGGA GCCGGTGAGC 840 GTGGGTCTCG CGGTATCATT GCAGCACTGGGGCCAGATGG TAAGCCCTCC CGTATCGTAG 900 TTATCTACAC GACGGGGAGT CAGGCAACTATGGATGAACG AAATAGACAG ATCGCTGAGA 960 TAGGTGCCTC ACTGATTAAG CATTGGTAACTGTCAGACCA AGTTTACTCA TATATACTTT 1020 AGATTGATTT AAAACTTCAT TTTTAATTTAAAAGGATCTA GGTGAAGATC CTTTTTGATA 1080 ATCTCATGAC CAAAATCCCT TAACGTGAGTTTTCGTTCCA CTGAGCGTCA GACCCCGTAG 1140 AAAAGATCAA AGGATCTTCT TGAGATCCTTTTTTTCTGCG CGTAATCTGC TGCTTGCAAA 1200 CAAAAAAACC ACCGCTACCA GCGGTGGTTTGTTTGCCGGA TCAAGAGCTA CCAACTCTTT 1260 TTCCGAAGGT AACTGGCTTC AGCAGAGCGCAGATACCAAA TACTGTCCTT CTAGTGTAGC 1320 CGTAGTTAGG CCACCACTTC AAGAACTCTGTAGCACCGCC TACATACCTC GCTCTGCTAA 1380 TCCTGTTACC AGTGGCTGCT GCCAGTGGCGATAAGTCGTG TCTTACCGGG TTGGACTCAA 1440 GACGATAGTT ACCGGATAAG GCGCAGCGGTCGGGCTGAAC GGGGGGTTCG TGCACACAGC 1500 CCAGCTTGGA GCGAACGACC TACACCGAACTGAGATACCT ACAGCGTGAG CTATGAGAAA 1560 GCGCCACGCT TCCCGAAGGG AGAAAGGCGGACAGGTATCC GGTAAGCGGC AGGGTCGGAA 1620 CAGGAGAGCG CACGAGGGAG CTTCCAGGGGGAAACGCCTG GTATCTTTAT AGTCCTGTCG 1680 GGTTTCGCCA CCTCTGACTT GAGCGTCGATTTTTGTGATG CTCGTCAGGG GGGCGGAGCC 1740 TATGGAAAAA CGCCAGCAAC GCGGCCTTTTTACGGTTCCT GGCCTTTTGC TGGCCTTTTG 1800 CTCACATGTT CTTTCCTGCG TTATCCCCTGATTCTGTGGA TAACCGTATT ACCGCCTTTG 1860 AGTGAGCTGA TACCGCTCGC CGCAGCCGAACGACCGAGCG CAGCGAGTCA GTGAGCGAGG 1920 AAGCGGAAGA GCGCCCAATA CGCAAACCGCCTCTCCCCGC GCGTTGGCCG ATTCATTAAT 1980 GCAGCTGGCA CGACAGGTTT CCCGACTGGAAAGCGGGCAG TGAGCGCAAC GCAATTAATG 2040 TGAGTTAGCT CACTCATTAG GCACCCCAGGCTTTACACTT TATGCTTCCG GCTCGTATGT 2100 TGTGTGGAAT TGTGAGCGGA TAACAATTTCACACAGGAAA CAGCTATGAC CATGATTACG 2160 CCAAGCGCGC AATTAACCCT CACTAAAGGGAACAAAAGCT GGAGCTCCAC CGCGGTGGCG 2220 GCCGCTCTAG CCCGGGCTAG AACTAGTGGATCCCCCAAAG CGGGGTTTGA ACAGGGTTTC 2280 GCTCAGGTTT GCCTGTGTCA TGGATGCAGCCTCCAGAATA CTTACTGGAA ACTATTGTAA 2340 CCCGCCTGAA GTTAAAAAGA ACAACGCCCGGCAGTGCCAG GCGTTGAAAA GATTAGCGAC 2400 CGGAGATTGG CGGGACGAAT ACGACGCCCATATCCCACGG CTGTTCAATC CAGGTATCTT 2460 GCGGGATATC AACAACATAG TCATCAACCAGCGGACGACC AGCCGGTTTT GCGAAGATGG 2520 TGACAAAGTG CGCTTTTGGA TACATTTCACGAATCGCAAC CGCAGTACCA CCGGTATCCA 2580 CCAGGTCATC AATAACGATG AAGCCTTCGCCATCGCCTTC TGCGCGTTTC AGCACTTTAA 2640 GCTCGCGCTG GTTGTCGTGA TCGTAGCTGGAAATACAAAC GGTATCGACA TGACGAATAC 2700 CCAGTTCACG CGCCAGTAAC GCACCCGGTACCAGACCGCC ACGGCTTACG GCAATAATGC 2760 CTTTCCATTG TTCAGAAGGC ATCAGTCGGCTTGCGAGTTT ACGTGCATGG ATCTGCAACA 2820 TGTCCCAGGT GACGATGTAT TTTTCGCTCATGTGAAGTGT CCCAGCCTGT TTATCTACGG 2880 CTTAAAAAGT GTTCGAGGGG AAAATAGGTTGCGCGAGATT ATAGAGATCT GGCGCACTAA 2940 AAACCAGTAT TTCACATGAG TCCGCGTCTTTTTACGCACT GCCTCTCCCT GACGCGGGAT 3000 AAAGTGGTAT TCTCAAACAT ATCTCGCAAGCCTGTCTTGT GTCCAAGCTT GGGGATCATC 3060 CGTCACTGTT CTTTATGATT CTACTTCCTTACCGTGCAAT AAATTAGAAT ATATTTTCTA 3120 CTTTTACGAG AAATTAATTA TTGTATTTATTATTTATGGG TGAAAAACTT ACTATAAAAA 3180 GCGGGTGGGT TTGGAATTAG TGATCAGTTTATGTATATCG CAACTACCGG CATATGGCTA 3240 TTCGACATCG AGAACATTAC CCACATGATAAGAGATTGTA TCAGTTTCGT AGTCTTGAGT 3300 ATTGGTATTA CTATATAGTA TATAGATGTCGAGGCGGTAC CCTTAAGTTG GGCTGCAGAA 3360 GCTTTTTTTT TTTTTTTTTT TTGGCATATAAATCGTTAAC GAATTCCATG GCCCGGGAAG 3420 GCCTCGGACC GGGCCCGGCC ATATAGGCCAGCGATACCGT CGCGGCCGCG ACCTCGAGGG 3480 GGGGCCCGGT ACCCAATTCG CCCTATAGTGAGTCGTATTA CGCGCGCTCA CTGGCCGTCG 3540 TTTTACAACG TCGTGACTGG GAAAACCCTGGCGTTACCCA ACTTAATCGC CTTGCAGCAC 3600 ATCCCCCTTT CGCCAGCTGG CGTAATAGCGAAGAGGCCCG CACCGATCGC CCTTCCCAAC 3660 AGTTGCGCAG CCTGAATGGC GAATGGAAATTGTAAGCGTT AATATTTTGT TAAAATTCGC 3720 GTTAAATTTT TGTTAAATCA GCTCATTTTTTAACCAATAG GCCGAAATCG GCAAAATCCC 3780 TTATAAATCA AAAGAATAGA CCGAGATAGGGTTGAGTGTT GTTCCAGTTT GGAACAAGAG 3840 TCCACTATTA AAGAACGTGG ACTCCAACGTCAAAGGGCGA AAAACCGTCT ATCAGGGCGA 3900 TGGCCCACTA CGTGAACCAT CACCCTAATCAAGTTTTTTG GGGTCGAGGT GCCGTAAAGC 3960 ACTAAATCGG AACCCTAAAG GGAGCCCCCGATTTAGAGCT TGACGGGGAA AGCCGGCGAA 4020 CGTGGCGAGA AAGGAAGGGA AGAAAGCGAAAGGAGCGGGC GCTAGGGCGC TGGCAAGTGT 4080 AGCGGTCACG CTGCGCGTAA CCACCACACCCGCCGCGCTT AATGCGCCGC TACAGGGCGC 4140 GTCAG 4145 (2) INFORMATION FOR SEQID NO:63: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4277 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: pS2gpt-P2 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:63: GTGGCACTTT TCGGGGAAAT GTGCGCGGAACCCCTATTTG TTTATTTTTC TAAATACATT 60 CAAATATGTA TCCGCTCATG AGACAATAACCCTGATAAAT GCTTCAATAA TATTGAAAAA 120 GGAAGAGTAT GAGTATTCAA CATTTCCGTGTCGCCCTTAT TCCCTTTTTT GCGGCATTTT 180 GCCTTCCTGT TTTTGCTCAC CCAGAAACGCTGGTGAAAGT AAAAGATGCT GAAGATCAGT 240 TGGGTGCACG AGTGGGTTAC ATCGAACTGGATCTCAACAG CGGTAAGATC CTTGAGAGTT 300 TTCGCCCCGA AGAACGTTTT CCAATGATGAGCACTTTTAA AGTTCTGCTA TGTGGCGCGG 360 TATTATCCCG TATTGACGCC GGGCAAGAGCAACTCGGTCG CCGCATACAC TATTCTCAGA 420 ATGACTTGGT TGAGTACTCA CCAGTCACAGAAAAGCATCT TACGGATGGC ATGACAGTAA 480 GAGAATTATG CAGTGCTGCC ATAACCATGAGTGATAACAC TGCGGCCAAC TTACTTCTGA 540 CAACGATCGG AGGACCGAAG GAGCTAACCGCTTTTTTGCA CAACATGGGG GATCATGTAA 600 CTCGCCTTGA TCGTTGGGAA CCGGAGCTGAATGAAGCCAT ACCAAACGAC GAGCGTGACA 660 CCACGATGCC TGTAGCAATG GCAACAACGTTGCGCAAACT ATTAACTGGC GAACTACTTA 720 CTCTAGCTTC CCGGCAACAA TTAATAGACTGGATGGAGGC GGATAAAGTT GCAGGACCAC 780 TTCTGCGCTC GGCCCTTCCG GCTGGCTGGTTTATTGCTGA TAAATCTGGA GCCGGTGAGC 840 GTGGGTCTCG CGGTATCATT GCAGCACTGGGGCCAGATGG TAAGCCCTCC CGTATCGTAG 900 TTATCTACAC GACGGGGAGT CAGGCAACTATGGATGAACG AAATAGACAG ATCGCTGAGA 960 TAGGTGCCTC ACTGATTAAG CATTGGTAACTGTCAGACCA AGTTTACTCA TATATACTTT 1020 AGATTGATTT AAAACTTCAT TTTTAATTTAAAAGGATCTA GGTGAAGATC CTTTTTGATA 1080 ATCTCATGAC CAAAATCCCT TAACGTGAGTTTTCGTTCCA CTGAGCGTCA GACCCCGTAG 1140 AAAAGATCAA AGGATCTTCT TGAGATCCTTTTTTTCTGCG CGTAATCTGC TGCTTGCAAA 1200 CAAAAAAACC ACCGCTACCA GCGGTGGTTTGTTTGCCGGA TCAAGAGCTA CCAACTCTTT 1260 TTCCGAAGGT AACTGGCTTC AGCAGAGCGCAGATACCAAA TACTGTCCTT CTAGTGTAGC 1320 CGTAGTTAGG CCACCACTTC AAGAACTCTGTAGCACCGCC TACATACCTC GCTCTGCTAA 1380 TCCTGTTACC AGTGGCTGCT GCCAGTGGCGATAAGTCGTG TCTTACCGGG TTGGACTCAA 1440 GACGATAGTT ACCGGATAAG GCGCAGCGGTCGGGCTGAAC GGGGGGTTCG TGCACACAGC 1500 CCAGCTTGGA GCGAACGACC TACACCGAACTGAGATACCT ACAGCGTGAG CTATGAGAAA 1560 GCGCCACGCT TCCCGAAGGG AGAAAGGCGGACAGGTATCC GGTAAGCGGC AGGGTCGGAA 1620 CAGGAGAGCG CACGAGGGAG CTTCCAGGGGGAAACGCCTG GTATCTTTAT AGTCCTGTCG 1680 GGTTTCGCCA CCTCTGACTT GAGCGTCGATTTTTGTGATG CTCGTCAGGG GGGCGGAGCC 1740 TATGGAAAAA CGCCAGCAAC GCGGCCTTTTTACGGTTCCT GGCCTTTTGC TGGCCTTTTG 1800 CTCACATGTT CTTTCCTGCG TTATCCCCTGATTCTGTGGA TAACCGTATT ACCGCCTTTG 1860 AGTGAGCTGA TACCGCTCGC CGCAGCCGAACGACCGAGCG CAGCGAGTCA GTGAGCGAGG 1920 AAGCGGAAGA GCGCCCAATA CGCAAACCGCCTCTCCCCGC GCGTTGGCCG ATTCATTAAT 1980 GCAGCTGGCA CGACAGGTTT CCCGACTGGAAAGCGGGCAG TGAGCGCAAC GCAATTAATG 2040 TGAGTTAGCT CACTCATTAG GCACCCCAGGCTTTACACTT TATGCTTCCG GCTCGTATGT 2100 TGTGTGGAAT TGTGAGCGGA TAACAATTTCACACAGGAAA CAGCTATGAC CATGATTACG 2160 CCAAGCGCGC AATTAACCCT CACTAAAGGGAACAAAAGCT GGAGCTCCAC CGCGGTGGCG 2220 GCCGCTCTAG CCCGGGCTAG AACTAGTGGATCCCCCAAAG CGGGGTTTGA ACAGGGTTTC 2280 GCTCAGGTTT GCCTGTGTCA TGGATGCAGCCTCCAGAATA CTTACTGGAA ACTATTGTAA 2340 CCCGCCTGAA GTTAAAAAGA ACAACGCCCGGCAGTGCCAG GCGTTGAAAA GATTAGCGAC 2400 CGGAGATTGG CGGGACGAAT ACGACGCCCATATCCCACGG CTGTTCAATC CAGGTATCTT 2460 GCGGGATATC AACAACATAG TCATCAACCAGCGGACGACC AGCCGGTTTT GCGAAGATGG 2520 TGACAAAGTG CGCTTTTGGA TACATTTCACGAATCGCAAC CGCAGTACCA CCGGTATCCA 2580 CCAGGTCATC AATAACGATG AAGCCTTCGCCATCGCCTTC TGCGCGTTTC AGCACTTTAA 2640 GCTCGCGCTG GTTGTCGTGA TCGTAGCTGGAAATACAAAC GGTATCGACA TGACGAATAC 2700 CCAGTTCACG CGCCAGTAAC GCACCCGGTACCAGACCGCC ACGGCTTACG GCAATAATGC 2760 CTTTCCATTG TTCAGAAGGC ATCAGTCGGCTTGCGAGTTT ACGTGCATGG ATCTGCAACA 2820 TGTCCCAGGT GACGATGTAT TTTTCGCTCATGTGAAGTGT CCCAGCCTGT TTATCTACGG 2880 CTTAAAAAGT GTTCGAGGGG AAAATAGGTTGCGCGAGATT ATAGAGATCT GGCGCACTAA 2940 AAACCAGTAT TTCACATGAG TCCGCGTCTTTTTACGCACT GCCTCTCCCT GACGCGGGAT 3000 AAAGTGGTAT TCTCAAACAT ATCTCGCAAGCCTGTCTTGT GTCCAAGCTT GGGGATCATC 3060 CGTCACTGTT CTTTATGATT CTACTTCCTTACCGTGCAAT AAATTAGAAT ATATTTTCTA 3120 CTTTTACGAG AAATTAATTA TTGTATTTATTATTTATGGG TGAAAAACTT ACTATAAAAA 3180 GCGGGTGGGT TTGGAATTAG TGATCAGTTTATGTATATCG CAACTACCGG CATATGGCTA 3240 TTCGACATCG AGAACATTAC CCACATGATAAGAGATTGTA TCAGTTTCGT AGTCTTGAGT 3300 ATTGGTATTA CTATATAGTA TATAGATGTCGAGGCGGTAC CCTTAAGTTG GGCTGCAGTT 3360 GTTAGAGCTT GGTATAGCGG ACAACTAAGTAATTGTAAAG AAGAAAACGA AACTATCAAA 3420 ACCGTTTATG AAATGATAGA AAAAAGAATATAAATAATCC TGTATTTTAG TTTAAGTAAC 3480 AGTAAAATAA TGAGTAGAAA ATACTATTTTTTATAGCCTA TAAATCGTTA ACGAATTCCA 3540 TGGCCCGGGA AGGCCTCGGA CCGGGCCCGGCCATATAGGC CAGCGATACC GTCGCGGCCG 3600 CGACCTCGAG GGGGGGCCCG GTACCCAATTCGCCCTATAG TGAGTCGTAT TACGCGCGCT 3660 CACTGGCCGT CGTTTTACAA CGTCGTGACTGGGAAAACCC TGGCGTTACC CAACTTAATC 3720 GCCTTGCAGC ACATCCCCCT TTCGCCAGCTGGCGTAATAG CGAAGAGGCC CGCACCGATC 3780 GCCCTTCCCA ACAGTTGCGC AGCCTGAATGGCGAATGGAA ATTGTAAGCG TTAATATTTT 3840 GTTAAAATTC GCGTTAAATT TTTGTTAAATCAGCTCATTT TTTAACCAAT AGGCCGAAAT 3900 CGGCAAAATC CCTTATAAAT CAAAAGAATAGACCGAGATA GGGTTGAGTG TTGTTCCAGT 3960 TTGGAACAAG AGTCCACTAT TAAAGAACGTGGACTCCAAC GTCAAAGGGC GAAAAACCGT 4020 CTATCAGGGC GATGGCCCAC TACGTGAACCATCACCCTAA TCAAGTTTTT TGGGGTCGAG 4080 GTGCCGTAAA GCACTAAATC GGAACCCTAAAGGGAGCCCC CGATTTAGAG CTTGACGGGG 4140 AAAGCCGGCG AACGTGGCGA GAAAGGAAGGGAAGAAAGCG AAAGGAGCGG GCGCTAGGGC 4200 GCTGGCAAGT GTAGCGGTCA CGCTGCGCGTAACCACCACA CCCGCCGCGC TTAATGCGCC 4260 GCTACAGGGC GCGTCAG 4277 (2)INFORMATION FOR SEQ ID NO:64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:4701 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pTZ-L2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: AGCGCCCAATACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTTTT 60 TCTGCGGCCGCGGCCTATAT GGCCCGGTCC GGTTAACTAC GTAGACGTCG AGGATTTCGC 120 GTGGGTCAATGCCGCGCCAG ATCCACATCA GACGGTTAAT CATGCGATAC CAGTGAGGGA 180 TGGTTTTACCATCAAGGGCC GACTGCACAG GCGGTTGTGC GCCGTGATTA AAGCGGCGGA 240 CTAGCGTCGAGGTTTCAGGA TGTTTAAAGC GGGGTTTGAA CAGGGTTTCG CTCAGGTTTG 300 CCTGTGTCATGGATGCAGCC TCCAGAATAC TTACTGGAAA CTATTGTAAC CCGCCTGAAG 360 TTAAAAAGAACAACGCCCGG CAGTGCCAGG CGTTGAAAAG ATTAGCGACC GGAGATTGGC 420 GGGACGAATACGACGCCCAT ATCCCACGGC TGTTCAATCC AGGTATCTTG CGGGATATCA 480 ACAACATAGTCATCAACCAG CGGACGACCA GCCGGTTTTG CGAAGATGGT GACAAAGTGC 540 GCTTTTGGATACATTTCACG AATCGCAACC GCAGTACCAC CGGTATCCAC CAGGTCATCA 600 ATAACGATGAAGCCTTCGCC ATCGCCTTCT GCGCGTTTCA GCACTTTAAG CTCGCGCTGG 660 TTGTCGTGATCGTAGCTGGA AATACAAACG GTATCGACAT GACGAATACC CAGTTCACGC 720 GCCAGTAACGCACCCGGTAC CAGACCGCCA CGGCTTACGG CAATAATGCC TTTCCATTGT 780 TCAGAAGGCATCAGTCGGCT TGCGAGTTTA CGTGCATGGA TCTGCAACAT GTCCCAGGTG 840 ACGATGTATTTTTCGCTCAT GTGAAGTGTC CCAGCCTGTT TATCTACGGC TTAAAAAGTG 900 TTCGAGGGGAAAATAGGTTG CGCGAGATTA TAGAGATCTG GCGCACTAAA AACCAGTATT 960 TCACATGAGTCCGCGTCTTT TTACGCACTG CCTCTCCCTG ACGCGGGATA AAGTGGTATT 1020 CTCAAACATATCTCGCAAGC CTGTCTTGTG TCCAAGCTTG GGGATCATCC GTCACTGTTC 1080 TTTATGATTCTACTTCCTTA CCGTGCAATA AATTAGAATA TATTTTCTAC TTTTACGAGA 1140 AATTAATTATTGTATTTATT ATTTATGGGT GAAAAACTTA CTATAAAAAG CGGGTGGGTT 1200 TGGAATTAGTGATCAGTTTA TGTATATCGC AACTACCGGC ATATGGCTAT TCGACATCGA 1260 GAACATTACCCACATGATAA GAGATTGTAT CAGTTTCGTA GTCTTGAGTA TTGGTATTAC 1320 TATATAGTATATNNNNNNGG TAACNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1380 NNNNNNNAGATCTCGATCCG GATATAGTTC CTCCTTTCAG CAAAAAACCC CTCAAGACCC 1440 GTTTAGAGGCCCCAAGGGGT TATGCTAGTT ATTGCTCANN NNNNNNNNGT CGACTTAATT 1500 AATTAGGCCTCTCGAGCTGC AGGGATCCAC TAGTGAGCTC CCCGGGGAAT TCCCATGGTA 1560 TTATCGTGTTTTTCAAAGGA AAAAAACGTC CCGTGGTTCG GGGGGCTCTN NNNNNNNNNN 1620 NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1680 NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1740 NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1800 NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1860 NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1920 NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1980 NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2040 NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2100 NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NCCGCTAGAG GGAAACCGTT GTGGTCTCCC 2160 TATAGTGAGTCGTATTAATT TCGCGGGATC GATCGATTAC GTAGTTAACG CGGCCGCGGC 2220 CTAGCCGGCCATAAAAATCT AGCTGGCGTA ATAGCGAAGA GGCCCGCACC GATCGCCCTT 2280 CCCAACAGTTGCGCAGCCTG AATGGCGAAT GGGAAATTGT AAACGTTAAT ATTTTGTTAA 2340 AATTCGCGTTAAATTTTTGT TAAATCAGCT CATTTTTTAA CCAATAGGCC GAAATCGGCA 2400 AAATCCCTTATAAATCAAAA GAATAGACCG AGATAGGGTT GAGTGTTGTT CCAGTTTGGA 2460 ACAAGAGTCCACTATTAAAG AACGTGGACT CCAACGTCAA AGGGCGAAAA ACCGTCTATC 2520 AGGGCGATGGCCCACTACGT GAACCATCAC CCTAATCAAG TTTTTTGGGG TCGAGGTGCC 2580 GTAAAGCACTAAATCGGAAC CCTAAAGGGA GCCCCCGATT TAGAGCTTGA CGGGGAAAGC 2640 CGGCGAACGTGGCGAGAAAG GAAGGGAAGA AAGCGAAAGG AGCGGGCGCT AGGGCGCTGG 2700 CAAGTGTAGCGGTCACGCTG CGCGTAACCA CCACACCCGC CGCGCTTAAT GCGCCGCTAC 2760 AGGGCGCGTCAGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT TGTTTATTTT 2820 TCTAAATACATTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA ATGCTTCAAT 2880 AATATTGAAAAAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT 2940 TTGCGGCATTTTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG 3000 CTGAAGATCAGTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA 3060 TCCTTGAGAGTTTTCGCCCC GAAGAACGTT TTCCAATGAT GAGCACTTTT AAAGTTCTGC 3120 TATGTGGCGCGGTATTATCC CGTGTTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC 3180 ACTATTCTCAGAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG 3240 GCATGACAGTAAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA 3300 ACTTACTTCTGACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG 3360 GGGATCATGTAACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG 3420 ACGAGCGTGACACCACGATG CCTGCAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG 3480 GCGAACTACTTACTCTAGCT TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG 3540 TTGCAGGACCACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG 3600 GAGCCGGTGAGCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT 3660 CCCGTATCGTAGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC 3720 AGATCGCTGAGATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC CAAGTTTACT 3780 CATATATACTTTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA 3840 TCCTTTTTGATAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT 3900 CAGACCCCGTAGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG CGCGTAATCT 3960 GCTGCTTGCAAACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG GATCAAGAGC 4020 TACCAACTCTTTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTCC 4080 TTCTAGTGTAGCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC 4140 TCGCTCTGCTAATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG 4200 GGTTGGACTCAAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT 4260 CGTGCACACAGCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG 4320 AGCATTGAGAAAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG 4380 GCAGGGTCGGAACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT 4440 ATAGTCCTGTCGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG 4500 GGGGGCGGAGCCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT 4560 GCTGGCCTTTTGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG GATAACCGTA 4620 TTACCGCCTTTGAGTGAGCT GATACCGCTC GCCGCAGCCG AACGACCGAG CGCAGCGAGT 4680 CAGTGAGCGAGGAAGCGGAA G 4701 (2) INFORMATION FOR SEQ ID NO:65: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 3878 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: pselP-gpt-L2 (xi) SEQUENCE DESCRIPTION: SEQID NO:65: AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAATGCAGCTTTT 60 TCTGCGGCCG CGGCCTATAT GGCCCGGTCC GGTTAACTAC GTAGACGTCGAGGATTTCGC 120 GTGGGTCAAT GCCGCGCCAG ATCCACATCA GACGGTTAAT CATGCGATACCAGTGAGGGA 180 TGGTTTTACC ATCAAGGGCC GACTGCACAG GCGGTTGTGC GCCGTGATTAAAGCGGCGGA 240 CTAGCGTCGA GGTTTCAGGA TGTTTAAAGC GGGGTTTGAA CAGGGTTTCGCTCAGGTTTG 300 CCTGTGTCAT GGATGCAGCC TCCAGAATAC TTACTGGAAA CTATTGTAACCCGCCTGAAG 360 TTAAAAAGAA CAACGCCCGG CAGTGCCAGG CGTTGAAAAG ATTAGCGACCGGAGATTGGC 420 GGGACGAATA CGACGCCCAT ATCCCACGGC TGTTCAATCC AGGTATCTTGCGGGATATCA 480 ACAACATAGT CATCAACCAG CGGACGACCA GCCGGTTTTG CGAAGATGGTGACAAAGTGC 540 GCTTTTGGAT ACATTTCACG AATCGCAACC GCAGTACCAC CGGTATCCACCAGGTCATCA 600 ATAACGATGA AGCCTTCGCC ATCGCCTTCT GCGCGTTTCA GCACTTTAAGCTCGCGCTGG 660 TTGTCGTGAT CGTAGCTGGA AATACAAACG GTATCGACAT GACGAATACCCAGTTCACGC 720 GCCAGTAACG CACCCGGTAC CAGACCGCCA CGGCTTACGG CAATAATGCCTTTCCATTGT 780 TCAGAAGGCA TCAGTCGGCT TGCGAGTTTA CGTGCATGGA TCTGCAACATGTCCCAGGTG 840 ACGATGTATT TTTCGCTCAT GTGAAGTGTC CCAGCCTGTT TATCTACGGCTTAAAAAGTG 900 TTCGAGGGGA AAATAGGTTG CGCGAGATTA TAGAGATCTG GCGCACTAAAAACCAGTATT 960 TCACATGAGT CCGCGTCTTT TTACGCACTG CCTCTCCCTG ACGCGGGATAAAGTGGTATT 1020 CTCAAACATA TCTCGCAAGC CTGTCTTGTG TCCAAGCTTG GGGATCATCCGTCACTGTTC 1080 TTTATGATTC TACTTCCTTA CCGTGCAATA AATTAGAATA TATTTTCTACTTTTACGAGA 1140 AATTAATTAT TGTATTTATT ATTTATGGGT GAAAAACTTA CTATAAAAAGCGGGTGGGTT 1200 TGGAATTAGT GATCAGTTTA TGTATATCGC AACTACCGGC ATATGATAAAAAGTCGACTT 1260 AATTAATTAG GCCTCTCGAG CTGCAGGGAT CCACTAGTGA GCTCCCCGGGGAATTCCCAT 1320 GGAGGCCTTA TTTATATTCC AAAAAAAAAA AATAAAATTT CAATTTTTATCGATTACGTA 1380 GTTAACGCGG CCGCGGCCTA GCCGGCCATA AAAATCTAGC TGGCGTAATAGCGAAGAGGC 1440 CCGCACCGAT CGCCCTTCCC AACAGTTGCG CAGCCTGAAT GGCGAATGGGAAATTGTAAA 1500 CGTTAATATT TTGTTAAAAT TCGCGTTAAA TTTTTGTTAA ATCAGCTCATTTTTTAACCA 1560 ATAGGCCGAA ATCGGCAAAA TCCCTTATAA ATCAAAAGAA TAGACCGAGATAGGGTTGAG 1620 TGTTGTTCCA GTTTGGAACA AGAGTCCACT ATTAAAGAAC GTGGACTCCAACGTCAAAGG 1680 GCGAAAAACC GTCTATCAGG GCGATGGCCC ACTACGTGAA CCATCACCCTAATCAAGTTT 1740 TTTGGGGTCG AGGTGCCGTA AAGCACTAAA TCGGAACCCT AAAGGGAGCCCCCGATTTAG 1800 AGCTTGACGG GGAAAGCCGG CGAACGTGGC GAGAAAGGAA GGGAAGAAAGCGAAAGGAGC 1860 GGGCGCTAGG GCGCTGGCAA GTGTAGCGGT CACGCTGCGC GTAACCACCACACCCGCCGC 1920 GCTTAATGCG CCGCTACAGG GCGCGTCAGG TGGCACTTTT CGGGGAAATGTGCGCGGAAC 1980 CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT CCGCTCATGAGACAATAACC 2040 CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAACATTTCCGTGT 2100 CGCCCTTATT CCCTTTTTTG CGGCATTTTG CCTTCCTGTT TTTGCTCACCCAGAAACGCT 2160 GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACATCGAACTGGA 2220 TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GAACGTTTTCCAATGATGAG 2280 CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT GTTGACGCCGGGCAAGAGCA 2340 ACTCGGTCGC CGCATACACT ATTCTCAGAA TGACTTGGTT GAGTACTCACCAGTCACAGA 2400 AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCATAACCATGAG 2460 TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA GGACCGAAGGAGCTAACCGC 2520 TTTTTTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT CGTTGGGAACCGGAGCTGAA 2580 TGAAGCCATA CCAAACGACG AGCGTGACAC CACGATGCCT GCAGCAATGGCAACAACGTT 2640 GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC CGGCAACAATTAATAGACTG 2700 GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGGCTGGCTGGTT 2760 TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTGCAGCACTGGG 2820 GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG ACGGGGAGTCAGGCAACTAT 2880 GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA CTGATTAAGCATTGGTAACT 2940 GTCAGACCAA GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATTTTTAATTTAA 3000 AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTTAACGTGAGTT 3060 TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTTGAGATCCTTT 3120 TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAGCGGTGGTTTG 3180 TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCAGCAGAGCGCA 3240 GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCAAGAACTCTGT 3300 AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTGCCAGTGGCGA 3360 TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGGCGCAGCGGTC 3420 GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCTACACCGAACT 3480 GAGATACCTA CAGCGTGAGC ATTGAGAAAG CGCCACGCTT CCCGAAGGGAGAAAGGCGGA 3540 CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGCTTCCAGGGGG 3600 AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTGAGCGTCGATT 3660 TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACGCGGCCTTTTT 3720 ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGTTATCCCCTGA 3780 TTCTGTGGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCCGCAGCCGAAC 3840 GACCGAGCGC AGCGAGTCAG TGAGCGAGGA AGCGGAAG 3878 (2)INFORMATION FOR SEQ ID NO:66: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:6474 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: pselP-gp160MN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: AGCGCCCAATACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTTTT 60 TCTGCGGCCGCGGCCTATAT GGCCCGGTCC GGTTAACTAC GTAGACGTCG AGGATTTCGC 120 GTGGGTCAATGCCGCGCCAG ATCCACATCA GACGGTTAAT CATGCGATAC CAGTGAGGGA 180 TGGTTTTACCATCAAGGGCC GACTGCACAG GCGGTTGTGC GCCGTGATTA AAGCGGCGGA 240 CTAGCGTCGAGGTTTCAGGA TGTTTAAAGC GGGGTTTGAA CAGGGTTTCG CTCAGGTTTG 300 CCTGTGTCATGGATGCAGCC TCCAGAATAC TTACTGGAAA CTATTGTAAC CCGCCTGAAG 360 TTAAAAAGAACAACGCCCGG CAGTGCCAGG CGTTGAAAAG ATTAGCGACC GGAGATTGGC 420 GGGACGAATACGACGCCCAT ATCCCACGGC TGTTCAATCC AGGTATCTTG CGGGATATCA 480 ACAACATAGTCATCAACCAG CGGACGACCA GCCGGTTTTG CGAAGATGGT GACAAAGTGC 540 GCTTTTGGATACATTTCACG AATCGCAACC GCAGTACCAC CGGTATCCAC CAGGTCATCA 600 ATAACGATGAAGCCTTCGCC ATCGCCTTCT GCGCGTTTCA GCACTTTAAG CTCGCGCTGG 660 TTGTCGTGATCGTAGCTGGA AATACAAACG GTATCGACAT GACGAATACC CAGTTCACGC 720 GCCAGTAACGCACCCGGTAC CAGACCGCCA CGGCTTACGG CAATAATGCC TTTCCATTGT 780 TCAGAAGGCATCAGTCGGCT TGCGAGTTTA CGTGCATGGA TCTGCAACAT GTCCCAGGTG 840 ACGATGTATTTTTCGCTCAT GTGAAGTGTC CCAGCCTGTT TATCTACGGC TTAAAAAGTG 900 TTCGAGGGGAAAATAGGTTG CGCGAGATTA TAGAGATCTG GCGCACTAAA AACCAGTATT 960 TCACATGAGTCCGCGTCTTT TTACGCACTG CCTCTCCCTG ACGCGGGATA AAGTGGTATT 1020 CTCAAACATATCTCGCAAGC CTGTCTTGTG TCCAAGCTTG GGGATCATCC GTCACTGTTC 1080 TTTATGATTCTACTTCCTTA CCGTGCAATA AATTAGAATA TATTTTCTAC TTTTACGAGA 1140 AATTAATTATTGTATTTATT ATTTATGGGT GAAAAACTTA CTATAAAAAG CGGGTGGGTT 1200 TGGAATTAGTGATCAGTTTA TGTATATCGC AACTACCGGC ATATGATAAA AAGTCGACTT 1260 AATTAATTAGGCTGGTTCAG CTCGTCTCAT TCTTTCCCTT ACAGTAGGCC ATCCAGTCAC 1320 ACGTTTTGACCATTTGCCAC CCATCTTATA GCAAAGCCCT TTCCAAGCCC TGTCTTATTC 1380 TTGTAGGTATGTGGAGAATA GCTCTACCAG CTCTTTGCAG TACTTCTATA ACCCTATCTG 1440 TCCCCTCAGCTACTGCTATA GCTGTGGCAT TAAGCAAGCT AACAGCACTA CTCTTTAGTT 1500 CCTGACTCCAATACTGTAGG AGATTCCACC AATATTTGAG GACTTCCCAC CCCCTGCGTC 1560 CCAGAAGTTCCACAATCCTC GCTGCAATCA AGAGTAAGTC TCTGTGGTGG TAGCTGAAGA 1620 GGAACAGGCTCCGCAGGTCG ACCCAGATAA TTGCTAAGAA TCCATGCACT AATCGACCGG 1680 ATGTGTCTCTGTCTCTCTCT CCACCTTCTT CTTCGATTCC TTCGGGCCTG TCGGGTCCCC 1740 TCGGAACTGGGGGGCGGGTC TGCAACGACA ATGGTGAGTA TCCCTGCCTA ACTCTATTCA 1800 CTATAGAAAGTACAGCAAAA ACTATTCTTA AACCTACCAA GCCTCCTACT ATCATTATGA 1860 ATATTTTTATATACCACAGC CAATTTGTTA TGTCAAACCA ATTCCACAAA CTTGCCCATT 1920 TATCCAATTCCAATAATTCT TGTTCATTCT TTTCTTGTTG GGTTTGCGAT TTTTCTAGTA 1980 ATGAGTATATTAAGCTTGTG TAATTGTCAA TTTCTCTTTC CCACTGCATC CAGGTCATGT 2040 TATTCCAAATATCATCCAGA GATTTATTAC TCCAACTAGC ATTCCAAGGC ACAGTAGTGG 2100 TGCAAATGAGTTTTCCAGAG CAACCCCAAA ACCCCAGGAG CTGTTGATCC TTTAGGTATC 2160 TTTCCACAGCCAGGACTCTT GCCTGGAGCT GCTTGATGCC CCAGACTGTG AGTTGCAACA 2220 TATGCTGTTGCGCCTCAATG GCCCTCAGCA AATTGTTCTG CTGTTGCACT ATACCAGACA 2280 ATAATAGTCTGGCCTGTACC GTCAGCGTCA CTGACGCTGC GCCCATAGTG CTTCCTGCTG 2340 CTCCTAAGAACCCAAGGAAC AGAGCTCCTA TCGCTGCTCT TTTTTCTCTC TGCACCACTC 2400 TTCTCTTTGCCTTGGTGGGT GCTACTCCTA ATGGTTCAAT TGTTACTACT TTATATTTAT 2460 ATAATTCACTTCTCCAATTG TCCCTCATAT CTCCTCCTCC AGGTCTGAAG ATCTCGGTGT 2520 CGTTCGTGTCCGTGTCCTTA CCACCATCTC TTGTTAATAG TAGCCCTGTA ATATTTGATG 2580 AACATCTAATTTGTCCTTCA ATGGGAGGGG CATACATTGC TTTTCCTACT TCCTGCCACA 2640 TGTTTATAATTTGTTTTATT TTGCATTGAA GTGTGATATT GTTATTTGAC CCTGTAGTAT 2700 TATTCCAAGTATTATTACCA TTCCAAGTAC TATTAAACAG TGGTGATGTA TTACAGTAGA 2760 AAAATTCCCCTCCACAATTA AAACTGTGCA TTACAATTTC TGGGTCCCCT CCTGAGGATT 2820 GATTAAAGACTATTGTTTTA TTCTTAAATT GTTCTTTTAA TTTGCTAACT ATCTGTCTTA 2880 AAGTGTCATTCCATTTTGCT CTACTAATGT TACAATGTGC TTGTCTTATA GTTCCTATTA 2940 TATTTTTTGTTGTATAAAAT GCTCTCCCTG GTCCTATATG TATCCTTTTT CTTTTATTGT 3000 AGTTGGGTCTTGTACAATTA ATTTGTACAG ATTCATTCAG ATGTACTATG ATGGTTTTAG 3060 CATTATCAGTGAAATTCTCA GATCTAATTA CTACCTCTTC TTCTGCTAGA CTGCCATTTA 3120 ACAGCAGTTGAGTTGATACT ACTGGCCTAA TTCCATGTGT ACATTGTACT GTGCTGACAT 3180 TTTTACATGATCCTTTTCCA CTGAACTTTT TATCGTTACA TTTTAGAATC GCAAAACCAG 3240 CCGGGGCACAATAGTGTATG GGAATTGGCT CAAAGGATAT CTTTGGACAA GCTTGTGTAA 3300 TGACTGAGGTATTACAACTT ATCAACCTAT AGCTGGTACT ATCATTATCT ATTGATACTA 3360 TATCAAGTTTATAAAGAAGT GCATATTCTT TCTGCATCTT ATCTCTTATG CTTGTGGTGA 3420 TATTGAAAGAGCAGTTTTTC ATTTCTCCTC CCTTTATTGT TCCCTCGCTA TTACTATTGT 3480 TATTAGCAGTACTATTATTG GTATTAGTAG TATTCCTCAA ATCAGTGCAA TTTAAAGTAA 3540 CACAGAGTGGGGTTAATTTT ACACATGGCT TTAGGCTTTG ATCCCATAAA CTGATTATAT 3600 CCTCATGCATCTGTTCTACC ATGTTATTTT TCCACATGTT AAAATTTTCT GTCACATTTA 3660 CCAATTCTACTTCTTGTGGG TTGGGGTCTG TGGGTACACA GGCTTGTGTG GCCCAAACAT 3720 TATGTACCTCTGTATCATAT GCTTTAGCAT CTGATGCACA AAATAGAGTG GTGGTTGCTT 3780 CTTTCCACACAGGTACCCCA TAATAGACTG TGACCCACAA TTTTTCTGTA GCACTACAGA 3840 TCATTAATAACCCAAGGAGC ATCGTGCCCC ATCCCCACCA GTGCTGATAA TTCCTCCTGA 3900 TCCCCTTCACGGCCATGGAG GCCTTATTTA TATTCCAAAA AAAAAAAATA AAATTTCAAT 3960 TTTTATCGATTACGTAGTTA ACGCGGCCGC GGCCTAGCCG GCCATAAAAA TCTAGCTGGC 4020 GTAATAGCGAAGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC CTGAATGGCG 4080 AATGGGAAATTGTAAACGTT AATATTTTGT TAAAATTCGC GTTAAATTTT TGTTAAATCA 4140 GCTCATTTTTTAACCAATAG GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA 4200 CCGAGATAGGGTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA AAGAACGTGG 4260 ACTCCAACGTCAAAGGGCGA AAAACCGTCT ATCAGGGCGA TGGCCCACTA CGTGAACCAT 4320 CACCCTAATCAAGTTTTTTG GGGTCGAGGT GCCGTAAAGC ACTAAATCGG AACCCTAAAG 4380 GGAGCCCCCGATTTAGAGCT TGACGGGGAA AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA 4440 AGAAAGCGAAAGGAGCGGGC GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA 4500 CCACCACACCCGCCGCGCTT AATGCGCCGC TACAGGGCGC GTCAGGTGGC ACTTTTCGGG 4560 GAAATGTGCGCGGAACCCCT ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC 4620 TCATGAGACAATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA 4680 TTCAACATTTCCGTGTCGCC CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG 4740 CTCACCCAGAAACGCTGGTG AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG 4800 GTTACATCGAACTGGATCTC AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC 4860 GTTTTCCAATGATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTGTTG 4920 ACGCCGGGCAAGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT 4980 ACTCACCAGTCACAGAAAAG CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG 5040 CTGCCATAACCATGAGTGAT AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGGAC 5100 CGAAGGAGCTAACCGCTTTT TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT 5160 GGGAACCGGAGCTGAATGAA GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGCAG 5220 CAATGGCAACAACGTTGCGC AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC 5280 AACAATTAATAGACTGGATG GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC 5340 TTCCGGCTGGCTGGTTTATT GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA 5400 TCATTGCAGCACTGGGGCCA GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG 5460 GGAGTCAGGCAACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA 5520 TTAAGCATTGGTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC 5580 TTCATTTTTAATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA 5640 TCCCTTAACGTGAGTTTTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT 5700 CTTCTTGAGATCCTTTTTTT CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC 5760 TACCAGCGGTGGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG 5820 GCTTCAGCAGAGCGCAGATA CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC 5880 ACTTCAAGAACTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG 5940 CTGCTGCCAGTGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG 6000 ATAAGGCGCAGCGGTCGGGC TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA 6060 CGACCTACACCGAACTGAGA TACCTACAGC GTGAGCATTG AGAAAGCGCC ACGCTTCCCG 6120 AAGGGAGAAAGGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA 6180 GGGAGCTTCCAGGGGGAAAC GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT 6240 GACTTGAGCGTCGATTTTTG TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA 6300 GCAACGCGGCCTTTTTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC 6360 CTGCGTTATCCCCTGATTCT GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG 6420 CTCGCCGCAGCCGAACGACC GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG GAAG 6474 (2) INFORMATIONFOR SEQ ID NO:67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6811 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION:Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE:pN2-gpta ProtS (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: GTGGCACTTTTCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT 60 CAAATATGTATCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 120 GGAAGAGTATGAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT GCGGCATTTT 180 GCCTTCCTGTTTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT 240 TGGGTGCACGAGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT 300 TTCGCCCCGAAGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA TGTGGCGCGG 360 TATTATCCCGTATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA 420 ATGACTTGGTTGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA 480 GAGAATTATGCAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA 540 CAACGATCGGAGGACCGAAG GAGCTAACCG CTTTTTTGCA CAACATGGGG GATCATGTAA 600 CTCGCCTTGATCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA 660 CCACGATGCCTGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA 720 CTCTAGCTTCCCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC 780 TTCTGCGCTCGGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC 840 GTGGGTCTCGCGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG 900 TTATCTACACGACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA 960 TAGGTGCCTCACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT 1020 AGATTGATTTAAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA 1080 ATCTCATGACCAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG 1140 AAAAGATCAAAGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA 1200 CAAAAAAACCACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT 1260 TTCCGAAGGTAACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC 1320 CGTAGTTAGGCCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA 1380 TCCTGTTACCAGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA 1440 GACGATAGTTACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC 1500 CCAGCTTGGAGCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA 1560 GCGCCACGCTTCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA 1620 CAGGAGAGCGCACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG 1680 GGTTTCGCCACCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC 1740 TATGGAAAAACGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG 1800 CTCACATGTTCTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG 1860 AGTGAGCTGATACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG 1920 AAGCGGAAGAGCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT 1980 GCAGCTGGCACGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG 2040 TGAGTTAGCTCACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT 2100 TGTGTGGAATTGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG 2160 CCAAGCGCGCAATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC CGCGGTGGCG 2220 GCCGCGTCGACAGAAAAATT AATTAATTAT GGCCTCTCGA GCTGCAGCTG CCAAGAAGAA 2280 GATTCCTGTGCTGCTCTCAG GAAAATATGT CCCACTTGTT TTCTAATTCA ATAAAGATAC 2340 TGGTTTAAATGTGAAGCCAC ACAAGAGAAA GATGAAGCCA AAGCTGGTCC CCCTGAGGAA 2400 TTGTTTTGAAATAAGGCATT AGGACCCTCC ATTCAATTCA TATTTAATAG ACCACCATCT 2460 CTTCTGCCTTCATCAGGAAA AAAACAAAAA CATAAACAAA ATAGTATCTG CCTATGATTA 2520 ATAGTATTTAATTACACGCA CTTTTGTTTG AGTTTACTTC CTTGCTTTCT GAAAAAAACA 2580 TAGGTATTTAGACACTAGTT CATGATGATA AAATTAAAAA TTTAGTTTTA CAAACAAAAA 2640 TTGAAACTGTCATTTGTAGG AAAAAAATTC AAATTTAAAA TTGTTATTTT TCACTATTCT 2700 TAGATAGCAAGAGAAGTAAG AATTTCTTTA CTGTGATTTA TATCACAACA GAATTTTTTT 2760 CCTTGACAAAGGACCTTTTA AAAATCCCAG GAAAGGACCA CAAAATAATC AAAGACTGCA 2820 CATTGTAAATAAAACCCTTC AGCTGTTATT GAAACATAAG TATAATTACA CACAAGGAAA 2880 AGGTATTATAAGCAGAGAAA AGATGCCTTA AGAATTCTTT GTCTTTTTCC AAACTGATGG 2940 ACATGAGTGAGCTCTAATAT CATTATGTTT AGAAATGGCT TCATCCAGAT CCAACTGTAC 3000 ACCATTAATATTCACTTCCA TGCAGCCATT ATAAAAGGCA TTCACTGGTG TGGCACTGAA 3060 TGGAACATCTGGAAGGCCAC CCAGGTATGT GGCCACTTTT GCTTTCATTG CTTTGTCCAA 3120 GACGGCAAGTTGTCTTTGAA GGTCTTCATG GGAGATGGTT TCTATTTTAA GTGGTGTCGA 3180 CAACTCCAGATTGTTTCTGT TGACTCTAAA TTCCAGATGA GATTGTTGAT CGGAACATAG 3240 ACTTAGGGCCTGTATCCGAT ATATTACAGT ATTTTCAACA GATAACAGAA TATCCTGTGA 3300 TTTTTCAGAGGTGGAGTCCA CCAAGGACAC AGCAAAGGGC ACTGTGTTGT TACCAGAAAC 3360 CAAGGCAAGCATAACACCAG TGCCCGTGGA TGGACGAATA TTCAAGGTCA CATTTACATG 3420 CCAACCCTCAGCACTGGATA CATTATTATA ATCTATGTGA AATTGAGCAA TTCCAGAACC 3480 AGGATAGTAGGAGCCCTTCT CCACAGTAAC CAGGCAATGC TTATTTTGTT TTTCTTGAAT 3540 AATTTCCTTTATTCCAGAAG CTCCTTGCTT CATCAAATTC CAGCTTCGTA TACATCCATC 3600 TAGACGAGGGTTAATCGGTT TAATGAGTTC ACTTTCCACT TTCCGAGGGA ATCCTGCAAA 3660 GTATACTTTGGTTTCCAGCA ATCCATTTTC CGGCTTAAAA AGGGGTCCAG GTTTATTTAT 3720 ATCCATCACAGCTTCTTTAG CTATTTTAAT GCTAATACTA TGTTCTAATT CTTCCACAGA 3780 CACCATATTCCATAGACCAT TATTAATAAC ATCACCTCCA GTTGTGATTT TGGATGTATG 3840 TTCATTCTTAAGCTGAACTT CAATCTTTCC ACCACGAAGT GCAATCAGGA GCCACGCTGA 3900 GTGATCGATAGATTCTGCGT ACAGTATCAC GCCTTCTGAA TCATATGTCC GGAAATCAAA 3960 TTCTGCTGAAAATCTGCTGA TTTCTGGCAA ACGAAATTTT AAATATAAAA CAACCCCTGC 4020 AAACTGCTCCGCCAAGTAAA GTAATTCATA CTTTGTGTCA AGGTTCAAGG GAAGGCACAC 4080 TGAAACAACCTCACAACTCT TCTGATCTTG GGCAAGTTTG AATCCTTTCT TCCCATCACA 4140 ATAGCAAGTGTAACCTCCAG GGTAATTGAC ACAAAGCTGA GCACACATGT TCTCAGAGCA 4200 TTCATCTATATCTTCACAAG ACTTTGATTT GAGATTATAT CTGTAGCCTT CGGGGCATTC 4260 ACATTCAAAATCTCCTGGGA TGTTCTTGCA CACAGCTGTG CCACAAATGC TTGGCTTCAA 4320 AGAGCATTCATCCACATCTT TACAATCTTT CTTATTTGAA AGCATAACAA AACCATTTTT 4380 ACAGGAACAGTGGTAACTTC CAGGTGTATT ATCACAAATT TGACTGCAAC CTCCATTTAT 4440 ATTTGAGGGATCTTTGCATT CATTTATGTC AAATTCACAC TTTTCTCCTT GCCAACCTGG 4500 TTTACAAGTGCAAGTAAAAG AAGCTTTTCC ATCTTTGCAG CTCATATATC CATCTTCATT 4560 GCATGGCAGAGGACTACACT GGTCTGGAAT GGCATTGACA CAGCTTCTTA GGTCAGGATA 4620 AGCATTAGTTGACTGACGTG CAGCAGTGAA TAACCCAGTT TGAAAAGAGC GAAGACAAAC 4680 TAAGTATTTTGGATAAAAAT AATCCGTTTC CGGGTCATTT TCAAAGACCT CCCTGGCTTC 4740 TTCTTTATTGCACAGTTCTT CGATGCATTC TCTTTCAAGA TTACCCTGTT TGGTTTCTTC 4800 AAGTAAAGAATTTGCACGAC GCTTCCTAAC CAGGACTTGT GAAGCCTGTT GCTTTGACAA 4860 AAAGTTTGCCTCTGAGACGG GAAGCACTAG GAGGAGACAC GCCAGCAACG CCCCGCAGCG 4920 CCCACCCAGGACCCCCATGG AGGCCTTATT TATATTCCAA AAAAAAAAAA TAAAATTTCA 4980 ATTTTTAGATCCCCCAACTT AAGGGTACCG CCTCGACATC TATATACTAT ATAGTAATAC 5040 CAATACTCAAGACTACGAAA CTGATACAAT CTCTTATCAT GTGGGTAATG TTCTCGATGT 5100 CGAATAGCCATATGCCGGTA GTTGCGATAT ACATAAACTG ATCACTAATT CCAAACCCAC 5160 CCGCTTTTTATAGTAAGTTT TTCACCCATA AATAATAAAT ACAATAATTA ATTTCTCGTA 5220 AAAGTAGAAAATATATTCTA ATTTATTGCA CGGTAAGGAA GTAGAATCAT AAAGAACAGT 5280 GACGGATGATCCCCAAGCTT GGACACAAGA CAGGCTTGCG AGATATGTTT GAGAATACCA 5340 CTTTATCCCGCGTCAGGGAG AGGCAGTGCG TAAAAAGACG CGGACTCATG TGAAATACTG 5400 GTTTTTAGTGCGCCAGATCT CTATAATCTC GCGCAACCTA TTTTCCCCTC GAACACTTTT 5460 TAAGCCGTAGATAAACAGGC TGGGACACTT CACATGAGCG AAAAATACAT CGTCACCTGG 5520 GACATGTTGCAGATCCATGC ACGTAAACTC GCAAGCCGAC TGATGCCTTC TGAACAATGG 5580 AAAGGCATTATTGCCGTAAG CCGTGGCGGT CTGGTACCGG GTGCGTTACT GGCGCGTGAA 5640 CTGGGTATTCGTCATGTCGA TACCGTTTGT ATTTCCAGCT ACGATCACGA CAACCAGCGC 5700 GAGCTTAAAGTGCTGAAACG CGCAGAAGGC GATGGCGAAG GCTTCATCGT TATTGATGAC 5760 CTGGTGGATACCGGTGGTAC TGCGGTTGCG ATTCGTGAAA TGTATCCAAA AGCGCACTTT 5820 GTCACCATCTTCGCAAAACC GGCTGGTCGT CCGCTGGTTG ATGACTATGT TGTTGATATC 5880 CCGCAAGATACCTGGATTGA ACAGCCGTGG GATATGGGCG TCGTATTCGT CCCGCCAATC 5940 TCCGGTCGCTAATCTTTTCA ACGCCTGGCA CTGCCGGGCG TTGTTCTTTT TAACTTCAGG 6000 CGGGTTACAATAGTTTCCAG TAAGTATTCT GGAGGCTGCA TCCATGACAC AGGCAAACCT 6060 GAGCGAAACCCTGTTCAAAC CCCGCTTTGG GCTGCAGGAA TTCGATATCA AGCTTATCGA 6120 TACCGTCGCGGCCGCGACCT CGAGGGGGGG CCCGGTACCC AATTCGCCCT ATAGTGAGTC 6180 GTATTACGCGCGCTCACTGG CCGTCGTTTT ACAACGTCGT GACTGGGAAA ACCCTGGCGT 6240 TACCCAACTTAATCGCCTTG CAGCACATCC CCCTTTCGCC AGCTGGCGTA ATAGCGAAGA 6300 GGCCCGCACCGATCGCCCTT CCCAACAGTT GCGCAGCCTG AATGGCGAAT GGAAATTGTA 6360 AGCGTTAATATTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC ATTTTTTAAC 6420 CAATAGGCCGAAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA GATAGGGTTG 6480 AGTGTTGTTCCAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC CAACGTCAAA 6540 GGGCGAAAAACCGTCTATCA GGGCGATGGC CCACTACGTG AACCATCACC CTAATCAAGT 6600 TTTTTGGGGTCGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG CCCCCGATTT 6660 AGAGCTTGACGGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA 6720 GCGGGCGCTAGGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC CACACCCGCC 6780 GCGCTTAATGCGCCGCTACA GGGCGCGTCA G 6811 (2) INFORMATION FOR SEQ ID NO:68: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: oProtS1 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:68: ACCCAGGACC GCCATGGCGA AGCGCGC 27 (2) INFORMATION FOR SEQ IDNO:69: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6926 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: pP2-gp160MN (xi)SEQUENCE DESCRIPTION: SEQ ID NO:69: GTGGCACTTT TCGGGGAAAT GTGCGCGGAACCCCTATTTG TTTATTTTTC TAAATACATT 60 CAAATATGTA TCCGCTCATG AGACAATAACCCTGATAAAT GCTTCAATAA TATTGAAAAA 120 GGAAGAGTAT GAGTATTCAA CATTTCCGTGTCGCCCTTAT TCCCTTTTTT GCGGCATTTT 180 GCCTTCCTGT TTTTGCTCAC CCAGAAACGCTGGTGAAAGT AAAAGATGCT GAAGATCAGT 240 TGGGTGCACG AGTGGGTTAC ATCGAACTGGATCTCAACAG CGGTAAGATC CTTGAGAGTT 300 TTCGCCCCGA AGAACGTTTT CCAATGATGAGCACTTTTAA AGTTCTGCTA TGTGGCGCGG 360 TATTATCCCG TATTGACGCC GGGCAAGAGCAACTCGGTCG CCGCATACAC TATTCTCAGA 420 ATGACTTGGT TGAGTACTCA CCAGTCACAGAAAAGCATCT TACGGATGGC ATGACAGTAA 480 GAGAATTATG CAGTGCTGCC ATAACCATGAGTGATAACAC TGCGGCCAAC TTACTTCTGA 540 CAACGATCGG AGGACCGAAG GAGCTAACCGCTTTTTTGCA CAACATGGGG GATCATGTAA 600 CTCGCCTTGA TCGTTGGGAA CCGGAGCTGAATGAAGCCAT ACCAAACGAC GAGCGTGACA 660 CCACGATGCC TGTAGCAATG GCAACAACGTTGCGCAAACT ATTAACTGGC GAACTACTTA 720 CTCTAGCTTC CCGGCAACAA TTAATAGACTGGATGGAGGC GGATAAAGTT GCAGGACCAC 780 TTCTGCGCTC GGCCCTTCCG GCTGGCTGGTTTATTGCTGA TAAATCTGGA GCCGGTGAGC 840 GTGGGTCTCG CGGTATCATT GCAGCACTGGGGCCAGATGG TAAGCCCTCC CGTATCGTAG 900 TTATCTACAC GACGGGGAGT CAGGCAACTATGGATGAACG AAATAGACAG ATCGCTGAGA 960 TAGGTGCCTC ACTGATTAAG CATTGGTAACTGTCAGACCA AGTTTACTCA TATATACTTT 1020 AGATTGATTT AAAACTTCAT TTTTAATTTAAAAGGATCTA GGTGAAGATC CTTTTTGATA 1080 ATCTCATGAC CAAAATCCCT TAACGTGAGTTTTCGTTCCA CTGAGCGTCA GACCCCGTAG 1140 AAAAGATCAA AGGATCTTCT TGAGATCCTTTTTTTCTGCG CGTAATCTGC TGCTTGCAAA 1200 CAAAAAAACC ACCGCTACCA GCGGTGGTTTGTTTGCCGGA TCAAGAGCTA CCAACTCTTT 1260 TTCCGAAGGT AACTGGCTTC AGCAGAGCGCAGATACCAAA TACTGTCCTT CTAGTGTAGC 1320 CGTAGTTAGG CCACCACTTC AAGAACTCTGTAGCACCGCC TACATACCTC GCTCTGCTAA 1380 TCCTGTTACC AGTGGCTGCT GCCAGTGGCGATAAGTCGTG TCTTACCGGG TTGGACTCAA 1440 GACGATAGTT ACCGGATAAG GCGCAGCGGTCGGGCTGAAC GGGGGGTTCG TGCACACAGC 1500 CCAGCTTGGA GCGAACGACC TACACCGAACTGAGATACCT ACAGCGTGAG CTATGAGAAA 1560 GCGCCACGCT TCCCGAAGGG AGAAAGGCGGACAGGTATCC GGTAAGCGGC AGGGTCGGAA 1620 CAGGAGAGCG CACGAGGGAG CTTCCAGGGGGAAACGCCTG GTATCTTTAT AGTCCTGTCG 1680 GGTTTCGCCA CCTCTGACTT GAGCGTCGATTTTTGTGATG CTCGTCAGGG GGGCGGAGCC 1740 TATGGAAAAA CGCCAGCAAC GCGGCCTTTTTACGGTTCCT GGCCTTTTGC TGGCCTTTTG 1800 CTCACATGTT CTTTCCTGCG TTATCCCCTGATTCTGTGGA TAACCGTATT ACCGCCTTTG 1860 AGTGAGCTGA TACCGCTCGC CGCAGCCGAACGACCGAGCG CAGCGAGTCA GTGAGCGAGG 1920 AAGCGGAAGA GCGCCCAATA CGCAAACCGCCTCTCCCCGC GCGTTGGCCG ATTCATTAAT 1980 GCAGCTGGCA CGACAGGTTT CCCGACTGGAAAGCGGGCAG TGAGCGCAAC GCAATTAATG 2040 TGAGTTAGCT CACTCATTAG GCACCCCAGGCTTTACACTT TATGCTTCCG GCTCGTATGT 2100 TGTGTGGAAT TGTGAGCGGA TAACAATTTCACACAGGAAA CAGCTATGAC CATGATTACG 2160 CCAAGCGCGC AATTAACCCT CACTAAAGGGAACAAAAGCT GGAGCTCCAC CGCGGTGGCG 2220 GCCGCTCTAG CCCGGGCTAG AACTAGTGGATCCCCCAAAG CGGGGTTTGA ACAGGGTTTC 2280 GCTCAGGTTT GCCTGTGTCA TGGATGCAGCCTCCAGAATA CTTACTGGAA ACTATTGTAA 2340 CCCGCCTGAA GTTAAAAAGA ACAACGCCCGGCAGTGCCAG GCGTTGAAAA GATTAGCGAC 2400 CGGAGATTGG CGGGACGAAT ACGACGCCCATATCCCACGG CTGTTCAATC CAGGTATCTT 2460 GCGGGATATC AACAACATAG TCATCAACCAGCGGACGACC AGCCGGTTTT GCGAAGATGG 2520 TGACAAAGTG CGCTTTTGGA TACATTTCACGAATCGCAAC CGCAGTACCA CCGGTATCCA 2580 CCAGGTCATC AATAACGATG AAGCCTTCGCCATCGCCTTC TGCGCGTTTC AGCACTTTAA 2640 GCTCGCGCTG GTTGTCGTGA TCGTAGCTGGAAATACAAAC GGTATCGACA TGACGAATAC 2700 CCAGTTCACG CGCCAGTAAC GCACCCGGTACCAGACCGCC ACGGCTTACG GCAATAATGC 2760 CTTTCCATTG TTCAGAAGGC ATCAGTCGGCTTGCGAGTTT ACGTGCATGG ATCTGCAACA 2820 TGTCCCAGGT GACGATGTAT TTTTCGCTCATGTGAAGTGT CCCAGCCTGT TTATCTACGG 2880 CTTAAAAAGT GTTCGAGGGG AAAATAGGTTGCGCGAGATT ATAGAGATCT GGCGCACTAA 2940 AAACCAGTAT TTCACATGAG TCCGCGTCTTTTTACGCACT GCCTCTCCCT GACGCGGGAT 3000 AAAGTGGTAT TCTCAAACAT ATCTCGCAAGCCTGTCTTGT GTCCAAGCTT GGGGATCATC 3060 CGTCACTGTT CTTTATGATT CTACTTCCTTACCGTGCAAT AAATTAGAAT ATATTTTCTA 3120 CTTTTACGAG AAATTAATTA TTGTATTTATTATTTATGGG TGAAAAACTT ACTATAAAAA 3180 GCGGGTGGGT TTGGAATTAG TGATCAGTTTATGTATATCG CAACTACCGG CATATGGCTA 3240 TTCGACATCG AGAACATTAC CCACATGATAAGAGATTGTA TCAGTTTCGT AGTCTTGAGT 3300 ATTGGTATTA CTATATAGTA TATAGATGTCGAGGCGGTAC CCTTAAGTTG GGCTGCAGTT 3360 GTTAGAGCTT GGTATAGCGG ACAACTAAGTAATTGTAAAG AAGAAAACGA AACTATCAAA 3420 ACCGTTTATG AAATGATAGA AAAAAGAATATAAATAATCC TGTATTTTAG TTTAAGTAAC 3480 AGTAAAATAA TGAGTAGAAA ATACTATTTTTTATAGCCTA TAAATCGTTC CTCATGAGAG 3540 TGAAGGGGAT CAGGAGGAAT TATCAGCACTGGTGGGGATG GGGCACGATG CTCCTTGGGT 3600 TATTAATGAT CTGTAGTGCT ACAGAAAAATTGTGGGTCAC AGTCTATTAT GGGGTACCTG 3660 TGTGGAAAGA AGCAACCACC ACTCTATTTTGTGCATCAGA TGCTAAAGCA TATGATACAG 3720 AGGTACATAA TGTTTGGGCC ACACAAGCCTGTGTACCCAC AGACCCCAAC CCACAAGAAG 3780 TAGAATTGGT AAATGTGACA GAAAATTTTAACATGTGGAA AAATAACATG GTAGAACAGA 3840 TGCATGAGGA TATAATCAGT TTATGGGATCAAAGCCTAAA GCCATGTGTA AAATTAACCC 3900 CACTCTGTGT TACTTTAAAT TGCACTGATTTGAGGAATAC TACTAATACC AATAATAGTA 3960 CTGCTAATAA CAATAGTAAT AGCGAGGGAACAATAAAGGG AGGAGAAATG AAAAACTGCT 4020 CTTTCAATAT CACCACAAGC ATAAGAGATAAGATGCAGAA AGAATATGCA CTTCTTTATA 4080 AACTTGATAT AGTATCAATA GATAATGATAGTACCAGCTA TAGGTTGATA AGTTGTAATA 4140 CCTCAGTCAT TACACAAGCT TGTCCAAAGATATCCTTTGA GCCAATTCCC ATACACTATT 4200 GTGCCCCGGC TGGTTTTGCG ATTCTAAAATGTAACGATAA AAAGTTCAGT GGAAAAGGAT 4260 CATGTAAAAA TGTCAGCACA GTACAATGTACACATGGAAT TAGGCCAGTA GTATCAACTC 4320 AACTGCTGTT AAATGGCAGT CTAGCAGAAGAAGAGGTAGT AATTAGATCT GAGAATTTCA 4380 CTGATAATGC TAAAACCATC ATAGTACATCTGAATGAATC TGTACAAATT AATTGTACAA 4440 GACCCAACTA CAATAAAAGA AAAAGGATACATATAGGACC AGGGAGAGCA TTTTATACAA 4500 CAAAAAATAT AATAGGAACT ATAAGACAAGCACATTGTAA CATTAGTAGA GCAAAATGGA 4560 ATGACACTTT AAGACAGATA GTTAGCAAATTAAAAGAACA ATTTAAGAAT AAAACAATAG 4620 TCTTTAATCA ATCCTCAGGA GGGGACCCAGAAATTGTAAT GCACAGTTTT AATTGTGGAG 4680 GGGAATTTTT CTACTGTAAT ACATCACCACTGTTTAATAG TACTTGGAAT GGTAATAATA 4740 CTTGGAATAA TACTACAGGG TCAAATAACAATATCACACT TCAATGCAAA ATAAAACAAA 4800 TTATAAACAT GTGGCAGGAA GTAGGAAAAGCAATGTATGC CCCTCCCATT GAAGGACAAA 4860 TTAGATGTTC ATCAAATATT ACAGGGCTACTATTAACAAG AGATGGTGGT AAGGACACGG 4920 ACACGAACGA CACCGAGATC TTCAGACCTGGAGGAGGAGA TATGAGGGAC AATTGGAGAA 4980 GTGAATTATA TAAATATAAA GTAGTAACAATTGAACCATT AGGAGTAGCA CCCACCAAGG 5040 CAAAGAGAAG AGTGGTGCAG AGAGAAAAAAGAGCAGCGAT AGGAGCTCTG TTCCTTGGGT 5100 TCTTAGGAGC AGCAGGAAGC ACTATGGGCGCAGCGTCAGT GACGCTGACG GTACAGGCCA 5160 GACTATTATT GTCTGGTATA GTGCAACAGCAGAACAATTT GCTGAGGGCC ATTGAGGCGC 5220 AACAGCATAT GTTGCAACTC ACAGTCTGGGGCATCAAGCA GCTCCAGGCA AGAGTCCTGG 5280 CTGTGGAAAG ATACCTAAAG GATCAACAGCTCCTGGGGTT TTGGGGTTGC TCTGGAAAAC 5340 TCATTTGCAC CACTACTGTG CCTTGGAATGCTAGTTGGAG TAATAAATCT CTGGATGATA 5400 TTTGGAATAA CATGACCTGG ATGCAGTGGGAAAGAGAAAT TGACAATTAC ACAAGCTTAA 5460 TATACTCATT ACTAGAAAAA TCGCAAACCCAACAAGAAAA GAATGAACAA GAATTATTGG 5520 AATTGGATAA ATGGGCAAGT TTGTGGAATTGGTTTGACAT AACAAATTGG CTGTGGTATA 5580 TAAAAATATT CATAATGATA GTAGGAGGCTTGGTAGGTTT AAGAATAGTT TTTGCTGTAC 5640 TTTCTATAGT GAATAGAGTT AGGCAGGGATACTCACCATT GTCGTTGCAG ACCCGCCCCC 5700 CAGTTCCGAG GGGACCCGAC AGGCCCGAAGGAATCGAAGA AGAAGGTGGA GAGAGAGACA 5760 GAGACACATC CGGTCGATTA GTGCATGGATTCTTAGCAAT TATCTGGGTC GACCTGCGGA 5820 GCCTGTTCCT CTTCAGCTAC CACCACAGAGACTTACTCTT GATTGCAGCG AGGATTGTGG 5880 AACTTCTGGG ACGCAGGGGG TGGGAAGTCCTCAAATATTG GTGGAATCTC CTACAGTATT 5940 GGAGTCAGGA ACTAAAGAGT AGTGCTGTTAGCTTGCTTAA TGCCACAGCT ATAGCAGTAG 6000 CTGAGGGGAC AGATAGGGTT ATAGAAGTACTGCAAAGAGC TGGTAGAGCT ATTCTCCACA 6060 TACCTACAAG AATAAGACAG GGCTTGGAAAGGGCTTTGCT ATAAGATGGG TGGCAAATGG 6120 TCAAAACGTG TGACTGGATG GCCTACTGTAAGGGAAAGAA TGAGACGAGC TGAACCAGAA 6180 CGAATTCCAT GGCCCGGGAA GGCCTCGGACCGGGCCCGGC CATATAGGCC AGCGATACCG 6240 TCGCGGCCGC GACCTCGAGG GGGGGCCCGGTACCCAATTC GCCCTATAGT GAGTCGTATT 6300 ACGCGCGCTC ACTGGCCGTC GTTTTACAACGTCGTGACTG GGAAAACCCT GGCGTTACCC 6360 AACTTAATCG CCTTGCAGCA CATCCCCCTTTCGCCAGCTG GCGTAATAGC GAAGAGGCCC 6420 GCACCGATCG CCCTTCCCAA CAGTTGCGCAGCCTGAATGG CGAATGGAAA TTGTAAGCGT 6480 TAATATTTTG TTAAAATTCG CGTTAAATTTTTGTTAAATC AGCTCATTTT TTAACCAATA 6540 GGCCGAAATC GGCAAAATCC CTTATAAATCAAAAGAATAG ACCGAGATAG GGTTGAGTGT 6600 TGTTCCAGTT TGGAACAAGA GTCCACTATTAAAGAACGTG GACTCCAACG TCAAAGGGCG 6660 AAAAACCGTC TATCAGGGCG ATGGCCCACTACGTGAACCA TCACCCTAAT CAAGTTTTTT 6720 GGGGTCGAGG TGCCGTAAAG CACTAAATCGGAACCCTAAA GGGAGCCCCC GATTTAGAGC 6780 TTGACGGGGA AAGCCGGCGA ACGTGGCGAGAAAGGAAGGG AAGAAAGCGA AAGGAGCGGG 6840 CGCTAGGGCG CTGGCAAGTG TAGCGGTCACGCTGCGCGTA ACCACCACAC CCGCCGCGCT 6900 TAATGCGCCG CTACAGGGCG CGTCAG 6926(2) INFORMATION FOR SEQ ID NO:70: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: selP promoter (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: TATGAGATCTAAAAATTGAA ATTTTATTTT TTTTTTTTGG AATATAAAT 49 (2) INFORMATION FOR SEQ IDNO:71: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: oFIX.1 (xi) SEQUENCE DESCRIPTION: SEQID NO:71: TCATGTTCAC GCGCTCCATG GCCGCGGCCG CACC 34 (2) INFORMATION FORSEQ ID NO:72: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5532 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNAoligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE: pN2gpta-FIX (xi)SEQUENCE DESCRIPTION: SEQ ID NO:72: GTGGCACTTT TCGGGGAAAT GTGCGCGGAACCCCTATTTG TTTATTTTTC TAAATACATT 60 CAAATATGTA TCCGCTCATG AGACAATAACCCTGATAAAT GCTTCAATAA TATTGAAAAA 120 GGAAGAGTAT GAGTATTCAA CATTTCCGTGTCGCCCTTAT TCCCTTTTTT GCGGCATTTT 180 GCCTTCCTGT TTTTGCTCAC CCAGAAACGCTGGTGAAAGT AAAAGATGCT GAAGATCAGT 240 TGGGTGCACG AGTGGGTTAC ATCGAACTGGATCTCAACAG CGGTAAGATC CTTGAGAGTT 300 TTCGCCCCGA AGAACGTTTT CCAATGATGAGCACTTTTAA AGTTCTGCTA TGTGGCGCGG 360 TATTATCCCG TATTGACGCC GGGCAAGAGCAACTCGGTCG CCGCATACAC TATTCTCAGA 420 ATGACTTGGT TGAGTACTCA CCAGTCACAGAAAAGCATCT TACGGATGGC ATGACAGTAA 480 GAGAATTATG CAGTGCTGCC ATAACCATGAGTGATAACAC TGCGGCCAAC TTACTTCTGA 540 CAACGATCGG AGGACCGAAG GAGCTAACCGCTTTTTTGCA CAACATGGGG GATCATGTAA 600 CTCGCCTTGA TCGTTGGGAA CCGGAGCTGAATGAAGCCAT ACCAAACGAC GAGCGTGACA 660 CCACGATGCC TGTAGCAATG GCAACAACGTTGCGCAAACT ATTAACTGGC GAACTACTTA 720 CTCTAGCTTC CCGGCAACAA TTAATAGACTGGATGGAGGC GGATAAAGTT GCAGGACCAC 780 TTCTGCGCTC GGCCCTTCCG GCTGGCTGGTTTATTGCTGA TAAATCTGGA GCCGGTGAGC 840 GTGGGTCTCG CGGTATCATT GCAGCACTGGGGCCAGATGG TAAGCCCTCC CGTATCGTAG 900 TTATCTACAC GACGGGGAGT CAGGCAACTATGGATGAACG AAATAGACAG ATCGCTGAGA 960 TAGGTGCCTC ACTGATTAAG CATTGGTAACTGTCAGACCA AGTTTACTCA TATATACTTT 1020 AGATTGATTT AAAACTTCAT TTTTAATTTAAAAGGATCTA GGTGAAGATC CTTTTTGATA 1080 ATCTCATGAC CAAAATCCCT TAACGTGAGTTTTCGTTCCA CTGAGCGTCA GACCCCGTAG 1140 AAAAGATCAA AGGATCTTCT TGAGATCCTTTTTTTCTGCG CGTAATCTGC TGCTTGCAAA 1200 CAAAAAAACC ACCGCTACCA GCGGTGGTTTGTTTGCCGGA TCAAGAGCTA CCAACTCTTT 1260 TTCCGAAGGT AACTGGCTTC AGCAGAGCGCAGATACCAAA TACTGTCCTT CTAGTGTAGC 1320 CGTAGTTAGG CCACCACTTC AAGAACTCTGTAGCACCGCC TACATACCTC GCTCTGCTAA 1380 TCCTGTTACC AGTGGCTGCT GCCAGTGGCGATAAGTCGTG TCTTACCGGG TTGGACTCAA 1440 GACGATAGTT ACCGGATAAG GCGCAGCGGTCGGGCTGAAC GGGGGGTTCG TGCACACAGC 1500 CCAGCTTGGA GCGAACGACC TACACCGAACTGAGATACCT ACAGCGTGAG CTATGAGAAA 1560 GCGCCACGCT TCCCGAAGGG AGAAAGGCGGACAGGTATCC GGTAAGCGGC AGGGTCGGAA 1620 CAGGAGAGCG CACGAGGGAG CTTCCAGGGGGAAACGCCTG GTATCTTTAT AGTCCTGTCG 1680 GGTTTCGCCA CCTCTGACTT GAGCGTCGATTTTTGTGATG CTCGTCAGGG GGGCGGAGCC 1740 TATGGAAAAA CGCCAGCAAC GCGGCCTTTTTACGGTTCCT GGCCTTTTGC TGGCCTTTTG 1800 CTCACATGTT CTTTCCTGCG TTATCCCCTGATTCTGTGGA TAACCGTATT ACCGCCTTTG 1860 AGTGAGCTGA TACCGCTCGC CGCAGCCGAACGACCGAGCG CAGCGAGTCA GTGAGCGAGG 1920 AAGCGGAAGA GCGCCCAATA CGCAAACCGCCTCTCCCCGC GCGTTGGCCG ATTCATTAAT 1980 GCAGCTGGCA CGACAGGTTT CCCGACTGGAAAGCGGGCAG TGAGCGCAAC GCAATTAATG 2040 TGAGTTAGCT CACTCATTAG GCACCCCAGGCTTTACACTT TATGCTTCCG GCTCGTATGT 2100 TGTGTGGAAT TGTGAGCGGA TAACAATTTCACACAGGAAA CAGCTATGAC CATGATTACG 2160 CCAAGCGCGC AATTAACCCT CACTAAAGGGAACAAAAGCT GGAGCTCCAC CGCGGTGGCG 2220 GCCGCTTGTT AATTTTCAAT TCCAATGAATTAACCTTGGA AATCCATCTT TCATTAAGTG 2280 AGCTTTGTTT TTTCCTTAAT CCAGTTGACATACCGGGATA CCTTGGTATA TATTCCATAT 2340 TTGCCTTTCA TTGCACACTC TTCACCCCAGCTAATAATTC CAGTTAAGAA ACTGGTCCCT 2400 TCCACTTCAG TAACATGGGG TCCCCCACTATCTCCTTGAC ATGAATCTCT ACCTCCTTCA 2460 TGGAAGCCAG CACAGAACAT GTTGTTATAGATGGTGAACT TTGTAGATCG AAGACATGTG 2520 GCTCGGTCAA CAAGTGGAAC TCTAAGGTACTGAAGAACTA AAGCTGATCT CCCTTTGTGG 2580 AAGACTCTTC CCCAGCCACT TACATAGCCAGATCCAAATT TGAGGAAGAT GTTCGTGTAT 2640 TCCTTGTCAG CAATGCAAAT AGGTGTAACGTAGCTGTTTA GCACTAAGGG TTCGTCCAGT 2700 TCCAGAAGGG CAATGTCATG GTTGTACTTATTAATAGCTG CATTGTAGTT GTGGTGAGGA 2760 ATAATTCGAA TCACATTTCG CTTTTGCTCTGTATGTTCTG TCTCCTCAAT ATTATGTTCA 2820 CCTGCGACAA CTGTAATTTT AACACCAGTTTCAACACAGT GGGCAGCAGT TACAATCCAT 2880 TTTTCATTAA CGATAGAGCC TCCACAGAATGCATCAACTT TACCATTCAA AACAACCTGC 2940 CAAGGGAATT GACCTGGTTT GGCATCTTCTCCACCAACAA CCCGAGTGAA GTCATTAAAT 3000 GATTGGGTGC TTTGAGTGAT GTTATCCAAAATGGTTTCAG CTTCAGTAGA ATTTACATAG 3060 TCCACATCAG GAAAAACAGT CTCAGCACGGGTGAGCTTAG AAGTTTGTGA AACAGAAACT 3120 CTTCCACATG GAAATGGCAC TGCTGGTTCACAGGACTTCT GGTTTTCTGC AAGTCGATAT 3180 CCCTCAGTAC AGGAGCAAAC CACCTTGTTATCAGCACTAT TTTTACAAAA CTGCTCGCAT 3240 CTGCCATTCT TAATGTTACA TGTTACATCTAATTCACAGT TCTTTCCTTC AAATCCAAAG 3300 GGACACCAAC ATTCATAGGA ATTAATGTCATCCTTGCAAC TGCCGCCATT TAAACATGGA 3360 TTGGACTCAC ACTGATCTCC ATCAACATACTGCTTCCAAA ATTCAGTTGT TCTTTCAGTG 3420 TTTTCAAAAA CTTCTCGTGC TTCTTCAAAACTACACTTTT CTTCCATACA TTCTCTCTCA 3480 AGGTTCCCTT GAACAAACTC TTCCAATTTACCTGAATTAT ACCTCTTTGG CCGATTCAGA 3540 ATTTTGTTGG CGTTTTCATG ATCAAGAAAAACTGTACATT CAGCACTGAG TAGATATCCT 3600 AAAAGGCAGA TGGTGATGAG GCCTGGTGATTCTGCCATGA TCATGTTCAC GCGCTCCATG 3660 GAGGCCTTAT TTATATTCCA AAAAAAAAAAATAAAATTTC AATTTTTAGA TCCCCCAACT 3720 TAAGGGTACC GCCTCGACAT CTATATACTATATAGTAATA CCAATACTCA AGACTACGAA 3780 ACTGATACAA TCTCTTATCA TGTGGGTAATGTTCTCGATG TCGAATAGCC ATATGCCGGT 3840 AGTTGCGATA TACATAAACT GATCACTAATTCCAAACCCA CCCGCTTTTT ATAGTAAGTT 3900 TTTCACCCAT AAATAATAAA TACAATAATTAATTTCTCGT AAAAGTAGAA AATATATTCT 3960 AATTTATTGC ACGGTAAGGA AGTAGAATCATAAAGAACAG TGACGGATGA TCCCCAAGCT 4020 TGGACACAAG ACAGGCTTGC GAGATATGTTTGAGAATACC ACTTTATCCC GCGTCAGGGA 4080 GAGGCAGTGC GTAAAAAGAC GCGGACTCATGTGAAATACT GGTTTTTAGT GCGCCAGATC 4140 TCTATAATCT CGCGCAACCT ATTTTCCCCTCGAACACTTT TTAAGCCGTA GATAAACAGG 4200 CTGGGACACT TCACATGAGC GAAAAATACATCGTCACCTG GGACATGTTG CAGATCCATG 4260 CACGTAAACT CGCAAGCCGA CTGATGCCTTCTGAACAATG GAAAGGCATT ATTGCCGTAA 4320 GCCGTGGCGG TCTGGTACCG GGTGCGTTACTGGCGCGTGA ACTGGGTATT CGTCATGTCG 4380 ATACCGTTTG TATTTCCAGC TACGATCACGACAACCAGCG CGAGCTTAAA GTGCTGAAAC 4440 GCGCAGAAGG CGATGGCGAA GGCTTCATCGTTATTGATGA CCTGGTGGAT ACCGGTGGTA 4500 CTGCGGTTGC GATTCGTGAA ATGTATCCAAAAGCGCACTT TGTCACCATC TTCGCAAAAC 4560 CGGCTGGTCG TCCGCTGGTT GATGACTATGTTGTTGATAT CCCGCAAGAT ACCTGGATTG 4620 AACAGCCGTG GGATATGGGC GTCGTATTCGTCCCGCCAAT CTCCGGTCGC TAATCTTTTC 4680 AACGCCTGGC ACTGCCGGGC GTTGTTCTTTTTAACTTCAG GCGGGTTACA ATAGTTTCCA 4740 GTAAGTATTC TGGAGGCTGC ATCCATGACACAGGCAAACC TGAGCGAAAC CCTGTTCAAA 4800 CCCCGCTTTG GGCTGCAGGA ATTCGATATCAAGCTTATCG ATACCGTCGC GGCCGCGACC 4860 TCGAGGGGGG GCCCGGTACC CAATTCGCCCTATAGTGAGT CGTATTACGC GCGCTCACTG 4920 GCCGTCGTTT TACAACGTCG TGACTGGGAAAACCCTGGCG TTACCCAACT TAATCGCCTT 4980 GCAGCACATC CCCCTTTCGC CAGCTGGCGTAATAGCGAAG AGGCCCGCAC CGATCGCCCT 5040 TCCCAACAGT TGCGCAGCCT GAATGGCGAATGGAAATTGT AAGCGTTAAT ATTTTGTTAA 5100 AATTCGCGTT AAATTTTTGT TAAATCAGCTCATTTTTTAA CCAATAGGCC GAAATCGGCA 5160 AAATCCCTTA TAAATCAAAA GAATAGACCGAGATAGGGTT GAGTGTTGTT CCAGTTTGGA 5220 ACAAGAGTCC ACTATTAAAG AACGTGGACTCCAACGTCAA AGGGCGAAAA ACCGTCTATC 5280 AGGGCGATGG CCCACTACGT GAACCATCACCCTAATCAAG TTTTTTGGGG TCGAGGTGCC 5340 GTAAAGCACT AAATCGGAAC CCTAAAGGGAGCCCCCGATT TAGAGCTTGA CGGGGAAAGC 5400 CGGCGAACGT GGCGAGAAAG GAAGGGAAGAAAGCGAAAGG AGCGGGCGCT AGGGCGCTGG 5460 CAAGTGTAGC GGTCACGCTG CGCGTAACCACCACACCCGC CGCGCTTAAT GCGCCGCTAC 5520 AGGGCGCGTC AG 5532 (2) INFORMATIONFOR SEQ ID NO:73: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION:Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE:wild-type gp160MN (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3..14(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: CA ATG AGA GTG AAG 14 Met ArgVal Lys 1 (2) INFORMATION FOR SEQ ID NO:74: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO:74: Met Arg Val Lys 1 (2) INFORMATION FOR SEQ ID NO:75: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: gp160 in vselP-gp160 virus (ix) FEATURE:(A) NAME/KEY: CDS (B) LOCATION: 3..14 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:75: CC ATG GCC GTG AAG 14 Met Ala Val Lys 1 (2) INFORMATION FOR SEQID NO:76: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO:76: Met Ala Val Lys 1 (2) INFORMATIONFOR SEQ ID NO:77: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION:Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B) CLONE:wild-type Protein S (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 4..18(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: GAA ATG AGG GTC CTG GGT 18 MetArg Val Leu Gly 1 5 (2) INFORMATION FOR SEQ ID NO:78: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO:78: Met Arg Val Leu Gly 1 5 (2) INFORMATION FOR SEQ ID NO:79:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other nucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide(vii) IMMEDIATE SOURCE: (B) CLONE: Protien S in the chimeras (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 4..18 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:79: GCC ATG GCG GTC CTG GGT 18 Met Ala Val LeuGly 1 5 (2) INFORMATION FOR SEQ ID NO:80: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: Met AlaVal Leu Gly 1 5 (2) INFORMATION FOR SEQ ID NO:81: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Othernucleic acid; (A) DESCRIPTION: Synthetic DNA oligonucleotide (vii)IMMEDIATE SOURCE: (B) CLONE: wild-type factor IX (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 3..17 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:81: TT ATG CAG CGC GTG AAC 17 Met Gln Arg Val Asn 1 5 (2) INFORMATIONFOR SEQ ID NO:82: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 aminoacids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: Met Gln Arg Val Asn 1 5(2) INFORMATION FOR SEQ ID NO:83: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A)DESCRIPTION: Synthetic DNA oligonucleotide (vii) IMMEDIATE SOURCE: (B)CLONE: factor IX vFIX#5 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:3..17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: CC ATG GAG CGC GTG AAC 17Met Glu Arg Val Asn 1 5 (2) INFORMATION FOR SEQ ID NO:84: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO:84: Met Glu Arg Val Asn 1 5 (2) INFORMATION FOR SEQ ID NO:85:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO:85: GGCCNNNNNG GCC 13 (2) INFORMATION FOR SEQ IDNO:86: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO:86: TAATTAATTA A 11 (2) INFORMATION FOR SEQ IDNO:87: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO:87: CTCGTAAAAA TTGAAAAACT ATTCTAATTT ATTGCACGGTACGTAC 46 (2) INFORMATION FOR SEQ ID NO:88: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 174 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO:88: ATGCATTTGT TAGAGCTTGG TATAGCGGAC AACTAAGTAA TTGTAAAGAAGAAAACGAAA 60 CTATCAAAAC CGTTTATGAA ATGATAGAAA AAAGAATATA AATAATCCTGTATTTTAGTT 120 TAAGTAACAG TAAAATAATG AGTAGAAAAT ACTATTTTTT ATAGCCTATAAATC 174 (2) INFORMATION FOR SEQ ID NO:89: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 234 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:ATGCATTTGT TAGAGCTTGG TATAGCGGAC AACTAAGTAA TTGTAAAGAA GAAAACGAAA 60CTATCAAAAC CGTTTATGAA ATGATAGAAA AAAGAATATA AATAATCCTG TATTTTAGTT 120TAAGTAACAG TAAAATAATG AGTAGAAAAT ACTATTTTTT ATAGCCTATA AATCGTTCTC 180GTAAAAATTG AAAAACTATT CTAATTTATT GCACGGTACG TACCATGGCC CGGG 234 (2)INFORMATION FOR SEQ ID NO:90: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: CTCGTAAAAATTGAAAAACT ATTCTAATTT ATTGCACGGT CGCGA 45 (2) INFORMATION FOR SEQ IDNO:91: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO:91: CATGGTACGT ACCGTGCAAT AAATTAGAAT AGTTTTTCAATTTTTACGAG 50 (2) INFORMATION FOR SEQ ID NO:92: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQID NO:92: GCTCCCGCAG GTACCGATGC AAATGGCCAC 30 (2) INFORMATION FOR SEQ IDNO:93: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO:93: GGGGAGAGAT CGAAAGTGAA TTTGACATAG C 31 (2)INFORMATION FOR SEQ ID NO:94: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: ACCATGGGTGCGAGAGCGTC GGTATTAAGC GGGGGAGAAT TAGAT 45 (2) INFORMATION FOR SEQ IDNO:95: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 51 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO:95: CGATCTAATT CTCCCCCGCT TAATACCGAC GCTCTCGCACCCATGGTAGC T 51

What is claimed is:
 1. A method for producing a protein employing amodified vaccinia viral expression system comprising the followingsteps: (a) providing a modified vaccinia virus containing a heterologousinsert encoding a protein, wherein said insert was molecularly cloneddirectly into the viral genome into a unique restriction endonucleasecleavage site; (b) infecting a vertebrate cell culture with the modifiedvaccinia virus from step (a); (c) culturing the infected cells underconditions resulting in expression of the protein; and, (d) isolatingthe protein produced by the infected cells.
 2. The method according toclaim 1, wherein the protein is selected from the group consisting ofHIV gp160, HIV Gag, and HIV Gag-Pol.
 3. The method according to claim 1,wherein the protein is selected from the group consisting ofprothrombin, Factor IX, Protein S, von Willebrand Factor,lys-plasminogen, and glu-plasminogen.
 4. The method according to claim1, wherein the restriction endonuclease is selected from the groupconsisting of NotI, SmaI, ApaI, and RsrII.
 5. A method for producing aprotein employing a modified fowlpox viral expression system comprisingthe following steps: (a) providing a modified fowlpox virus containing aheterologous insert encoding a protein, wherein said insert wasmolecularly cloned directly into the viral genome into a uniquerestriction endonuclease cleavage site recognized by a restrictionendonuclease selected from the group consisting of NotI, SmaI, ApaI, andRsrII; (b) infecting a vertebrate cell culture with the modifiedvaccinia virus from step (a); (c) culturing the infected cells underconditions resulting in expression of the protein; and, (d) isolatingthe protein produced by the infected cells.
 6. The method according toclaim 5, wherein the protein is selected from the group consisting ofHIV gp160, HIV Gag, and HIV Gag-Pol.
 7. The method according to claim 5,wherein the protein is selected from the group consisting ofprothrombin, Factor IX, Protein S, von Willebrand Factor,lys-plasminogen, and glu-plasminogen.
 8. A method for producing aprotein employing a modified vaccinia viral expression system comprisingthe following steps: (a) infecting cells with a modified vaccinia viruscontaining a heterologous insert encoding a protein, wherein said insertwas molecularly cloned directly into the viral genome into a uniquerestriction endonuclease cleavage site; (b) culturing the infected cellsfrom step (a) under conditions resulting in expression of the protein.9. The method according to claim 8, further comprising isolating theprotein produced by the infected cells of step (b).
 10. The methodaccording to claim 8, wherein the protein is selected from the groupconsisting of HIV gp160, HIV Gag, and HIV Gag-Pol.
 11. The methodaccording to claim 8, wherein the protein is a human blood protein. 12.The method according to claim 11, wherein the human blood protein isselected from the group consisting of prothrombin, Factor IX, Protein S,von Willebrand Factor, lys-plasminogen, and glu-plasminogen.
 13. Amethod for producing a protein employing a modified fowlpox viralexpression system comprising the following steps: (a) infecting cellswith a modified fowlpox virus containing a heterologous insert encodinga protein, wherein said insert was molecularly cloned directly into theviral genome into a unique restriction endonuclease cleavage siterecognized by a restriction endonuclease selected from the groupconsisting of NotI, SmaI, ApaI, and RsrII; and, (b) culturing theinfected cells from step (a) under conditions resulting in expression ofthe protein.
 14. The method according to claim 13, further comprisingisolating the protein produced by the infected cells of step (b). 15.The method according to claim 13, wherein the protein is selected fromthe group consisting of HIV gp160, HIV Gag, and HIV Gag-Pol.
 16. Themethod according to claim 13, wherein the protein is a human bloodprotein.
 17. The method according to claim 16, wherein the human bloodprotein is selected from the group consisting of prothrombin, Factor IX,Protein S, von Willebrand Factor, lys-plasminogen, and glu-plasminogen.